Compositions and Methods for Inhibiting Expression of Eg5 Gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Eg5 gene (Eg5 gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the Eg5 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by Eg5 expression and the expression of the Eg5 gene using the pharmaceutical composition; and methods for inhibiting the expression of the Eg5 gene in a cell.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/797,176, filed Mar. 12, 2013 (allowed), which is a continuation of U.S. application Ser. No. 13/165,568, filed Jun. 21, 2011 (abandoned), which is a continuation of U.S. application Ser. No. 12/754,110, filed Apr. 5, 2010 (abandoned), which is a divisional of U.S. application Ser. No. 11/694,215, filed Mar. 30, 2007 (now U.S. Pat. No. 7,718,629, issued May 18, 2010) all which claim the benefit of U.S. Provisional Application No. 60/787,762, filed Mar. 31, 2006, and U.S. Provisional Application No. 60/870,259, filed Dec. 15, 2006. All prior applications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 16, 2015, is named 28983_US_CRF_sequencelisting.txt, and is 361,411 bytes in size.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the Eg5 gene and the use of the dsRNA to treat pathological processes mediated by Eg5 expression, such as cancer, alone or in combination with a dsRNA targeting vascular endothelian growth factor (VEGF).

BACKGROUND OF THE INVENTION

The maintenance of cell populations within an organism is governed by the cellular processes of cell division and programmed cell death. Within normal cells, the cellular events associated with the initiation and completion of each process is highly regulated. In proliferative disease such as cancer, one or both of these processes may be perturbed. For example, a cancer cell may have lost its regulation (checkpoint control) of the cell division cycle through either the overexpression of a positive regulator or the loss of a negative regulator, perhaps by mutation.

Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator. Hence, there is a need to develop new chemotherapeutic drugs that will restore the processes of checkpoint control and programmed cell death to cancerous cells.

One approach to the treatment of human cancers is to target a protein that is essential for cell cycle progression. In order for the cell cycle to proceed from one phase to the next, certain prerequisite events must be completed. There are checkpoints within the cell cycle that enforce the proper order of events and phases. One such checkpoint is the spindle checkpoint that occurs during the metaphase stage of mitosis. Small molecules that target proteins with essential functions in mitosis may initiate the spindle checkpoint to arrest cells in mitosis. Of the small molecules that arrest cells in mitosis, those which display anti-tumor activity in the clinic also induce apoptosis, the morphological changes associated with programmed cell death. An effective chemotherapeutic for the treatment of cancer may thus be one which induces checkpoint control and programmed cell death. Unfortunately, there are few compounds available for controlling these processes within the cell. Most compounds known to cause mitotic arrest and apoptosis act as tubulin binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle thereby causing mitotic arrest. Because most of these compounds specifically target the tubulin protein which is a component of all microtubules, they may also affect one or more of the numerous normal cellular processes in which microtubules have a role. Hence, there is also a need for small molecules that more specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins that are localized to the mitotic spindle and known to be required for formation and/or function of the bipolar mitotic spindle. Recently, there was a report of a small molecule that disturbs bipolarity of the mitotic spindle (Mayer, T. U. et. al. 1999. Science 286(5441) 971-4, herein incorporated by reference). More specifically, the small molecule induced the formation of an aberrant mitotic spindle wherein a monoastral array of microtubules emanated from a central pair of centrosomes, with chromosomes attached to the distal ends of the microtubules. The small molecule was dubbed “monastrol” after the monoastral array. This monoastral array phenotype had been previously observed in mitotic cells that were immunodepleted of the Eg5 motor protein. This distinctive monoastral array phenotype facilitated identification of monastrol as a potential inhibitor of Eg5. Indeed, monastrol was further shown to inhibit the Eg5 motor-driven motility of microtubules in an in vitro assay. The Eg5 inhibitor monastrol had no apparent effect upon the related kinesin motor or upon the motor(s) responsible for golgi apparatus movement within the cell. Cells that display the monoastral array phenotype either through immunodepletion of Eg5 or monastrol inhibition of Eg5 arrest in M-phase of the cell cycle. However, the mitotic arrest induced by either immunodepletion or inhibition of Eg5 is transient (Kapoor, T. M., 2000. J Cell Biol 150(5) 975-80). Both the monoastral array phenotype and the cell cycle arrest in mitosis induced by monastrol are reversible. Cells recover to form a normal bipolar mitotic spindle, to complete mitosis and to proceed through the cell cycle and normal cell proliferation. These data suggest that a small molecule inhibitor of Eg5 which induced a transient mitotic arrest may not be effective for the treatment of cancer cell proliferation. Nonetheless, the discovery that monastrol causes mitotic arrest is intriguing and hence there is a need to further study and identify compounds which can be used to modulate the Eg5 motor protein in a manner that would be effective in the treatment of human cancers. There is also a need to explore the use of these compounds in combination with other antineoplastic agents.

VEGF (also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a wide variety of tissues, and its overexpression or aberrant expression can result in a variety disorders, including cancers and retinal disorders such as age-related macular degeneration and other angiogenic disorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in the treatment of pathological processes mediated by Eg5 expression, there remains a need for an agent that can selectively and efficiently silence the Eg5 gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target Eg5 gene for use in treating pathological processes mediated by Eg5 expression.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Eg5 gene in a cell or mammal using such dsRNA, alone or in combination with a dsRNA targeting VEGF. The invention also provides compositions and methods for treating pathological conditions and diseases caused by the expression of the Eg5 gene, such as in cancer. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the Eg5 gene.

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 gene. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding Eg5, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing the Eg5, inhibits the expression of the Eg5 gene by at least 40%.

For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such modified sequence will be based on a first sequence of said dsRNA selected from the group consisting of the sense sequences of Tables 1-3 and a second sequence selected from the group consisting of the antisense sequences of Tables 1-3.

In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is generally a mammalian cell, such as a human cell.

In another embodiment, the invention provides a pharmaceutical composition for inhibiting the expression of the Eg5 gene in an organism, generally a human subject, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier or delivery vehicle.

In another embodiment, the invention provides a method for inhibiting the expression of the Eg5 gene in a cell, comprising the following steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid         (dsRNA), wherein the dsRNA comprises at least two sequences that         are complementary to each other. The dsRNA comprises a sense         strand comprising a first sequence and an antisense strand         comprising a second sequence. The antisense strand comprises a         region of complementarity which is substantially complementary         to at least a part of a mRNA encoding Eg5, and wherein the         region of complementarity is less than 30 nucleotides in length,         generally 19-24 nucleotides in length, and wherein the dsRNA,         upon contact with a cell expressing the Eg5, inhibits expression         of the Eg5 gene by at least 40%; and     -   (b) maintaining the cell produced in step (a) for a time         sufficient to obtain degradation of the mRNA transcript of the         Eg5 gene, thereby inhibiting expression of the Eg5 gene in the         cell.

In another embodiment, the invention provides methods for treating, preventing or managing pathological processes mediated by Eg5 expression, e.g. cancer, comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.

In another embodiment, the invention provides vectors for inhibiting the expression of the Eg5 gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of the Eg5 gene in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.

In a further embodiment, the invention provides the Eg5 dsRNA and the uses thereof as described above in combination with a second dsRNA targeting the VEGF mRNA. A combination of a dsRNA targeting Eg5 and a second dsRNA targeting VEGF provides complementary and synergiatic activity for treating hyperproliferative discords, particularly hepatic carcinoma.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Eg5 gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of the Eg5 gene using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The invention further provides this dsRNA in combination with a second dsRNA that inhibits the expression of the VEGF gene.

The dsRNAs of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the Eg5 gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and or maintenance of cancer cells in mammals. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the Eg5 gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by Eg5 expression, e.g. cancer, by targeting a gene involved in mitotic division.

The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of the Eg5 gene, as well as compositions and methods for treating diseases and disorders caused by the expression of Eg5, such as cancer, alone or in combination with a second dsRNA targeting the VEGF gene. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the Eg5 gene, together with a pharmaceutically acceptable carrier. As discussed above, such compositions can further include a second dsRNA targeting VEGF.

Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Eg5 gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Eg5 gene. The invention further provides the above pharmaceutical compositions further containing a second dsRNA designed to inhibit the expression of VEGF.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein, “Eg5” refers to the human kinesin family member 11, which is also known as KIF11, Eg5, HKSP, KNSL1 or TRIPS. Eg5 sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeq ID number:NM_(—)004523.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the Eg5 gene, including mRNA that is a product of RNA processing of a primary transcription product.

As used herein, VEGF, also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene contains 8 exons that express a 189-amino acid protein isoform. A 165-amino acid isoform lacks the residues encoded by exon 6, whereas a 121-amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7. VEGF can act on endothelial cells by binding to an endothelial tyrosine kinase receptor, such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved in endothelial cell differentiation and vasculogenesis. A third receptor, VEGFR-3 has been implicated in lymphogenesis.

The various isoforms have different biologic activities and clinical implications. For example, VEGF145 induces angiogenesis and like VEGF189 (but unlike VEGF165) VEGF145 binds efficiently to the extracellular matrix by a mechanism that is not dependent on extracellular matrix-associated heparin sulfates. VEGF displays activity as an endothelial cell mitogen and chemoattractant in vitro and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a wide variety of cancer cell types and promotes the growth of tumors by inducing the development of tumor-associated vasculature Inhibition of VEGF function has been shown to limit both the growth of primary experimental tumors as well as the incidence of metastases in immunocompromised mice. Various dsRNAs directed to VEGF are described in co-pending U.S. Ser. No. 11/078,073 and 11/340,080, herein incorporated by reference).

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding Eg5). For example, a polynucleotide is complementary to at least a part of a Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding Eg5.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 ‘-end of one strand and the 5’ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as they refer to the Eg5 gene, herein refer to the at least partial suppression of the expression of the Eg5 gene, as manifested by a reduction of the amount of mRNA transcribed from the Eg5 gene which may be isolated from a first cell or group of cells in which the Eg5 gene is transcribed and which has or have been treated such that the expression of the Eg5 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Eg5 gene transcription, e.g. the amount of protein encoded by the Eg5 gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis. In principle, Eg5 gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of the Eg5 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the Eg5 gene (or VEGF gene) is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, the Eg5 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the Eg5 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. Tables 1-3 provides values for inhibition of expression using various Eg5 dsRNA molecules at various concentrations.

As used herein in the context of Eg5 expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by Eg5 expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by Eg5 expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as the slowing and progression of hepatic carcinoma.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by Eg5 expression or an overt symptom of pathological processes mediated by Eg5 expression (alone or in combination with VEGF expression). The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by Eg5 expression, the patient's history and age, the stage of pathological processes mediated by Eg5 expression, and the administration of other anti-pathological processes mediated by Eg5 expression agents.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 gene (alone or incombinaton with a second dsRNA for inhibiting the expression of VEGF) in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Eg5 gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said Eg5 gene, inhibits the expression of said Eg5 gene by at least 40%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Eg5 gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the Eg5 gene is the human Eg5 gene. In specific embodiments, the antisense strand of the dsRNA comprises the sense sequences of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1-3 can readily be determined using the target sequence and the flanking Eg5 sequence. In embodiments using a second dsRNA targeting VEGF, such agents are exemplified in the Examples and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.

The dsRNA will comprise at least two nucleotide sequence selected from the groups of sequences provided in Tables 1-3. One of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of the Eg5 gene. As such, the dsRNA will comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1-3 and the second oligonucleotide is described as the antisense strand in Tables 1-3

The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1-3, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1-3 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1-3, and differing in their ability to inhibit the expression of the Eg5 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1-3 can readily be made using the Eg5 sequence and the target sequence provided.

In addition, the RNAi agents provided in Tables 1-3 identify a site in the Eg5 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1-3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the Eg5 gene. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target Eg5 gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1-3.

The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the Eg5 gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Eg5 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Eg5 gene is important, especially if the particular region of complementarity in the Eg5 gene is known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference

Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)—O]_(m)CH₃, O(CH₂)—OCH₃, O(CH₂)—NH₂, O(CH₂)—CH₃, O(CH₂)—ONH₂, and O(CH₂)—ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.

dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the Eg5 gene, such as pathological processes mediated by Eg5 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.

In another embodiment, such compositions will further comprise a second dsRNA that inhibits VEGF expression. dsRNA directed to VEGF are described in the Examples and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080.

The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of the Eg5 gene (and VEGF expression when a second dsRNA is included). In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by Eg5 expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the dsRNAs of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which dsRNAs of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N. Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G.sub.M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an dsRNA RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNA dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by Eg5 expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of the Eg5 Gene

The invention relates in particular to the use of a dsRNA or a pharmaceutical composition prepared therefrom for the treatment of cancer, e.g., for inhibiting tumor growth and tumor metastasis. For example, the dsRNA or a pharmaceutical composition prepared therefrom may be used for the treatment of solid tumors, like breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment of skin cancer, like melanoma, for the treatment of lymphomas and blood cancer. The invention further relates to the use of an dsRNA according to the invention or a pharmaceutical composition prepared therefrom for inhibiting eg5 expression and/or for inhibiting accumulation of ascites fluid and pleural effusion in different types of cancer, e.g., breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, melanoma, lymphomas and blood cancer. Owing to the inhibitory effect on eg5 expression, an dsRNA according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

The invention furthermore relates to the use of an dsRNA or a pharmaceutical composition thereof, e.g., for treating cancer or for preventing tumor metastasis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen. Other embodiments include the use of a second dsRNA used to inhibit the expression of VEGF.

The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent, or another dsRNA used to inhibit the expression of VEGF. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.

Methods for Inhibiting Expression of the Eg5 Gene

In yet another aspect, the invention provides a method for inhibiting the expression of the Eg5 gene in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target Eg5 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target Eg5 gene. Compositions and methods for inhibiting the expression of these Eg5 genes using dsRNAs can be performed as described elsewhere herein.

In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the Eg5 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Gene Walking of the Eg5 Gene

Initial Screening Set

siRNA design was carried out to identify siRNAs targeting Eg5 (also known as KIF11, HSKP, KNSL1 and TRIPS). Human mRNA sequences to Eg5, RefSeq ID number:NM_(—)004523, was used.

siRNA duplexes cross-reactive to human and mouse Eg5 were designed. Twenty-four duplexes were synthesized for screening. (Table 1).

Expanded Screening Set

A second screening set was defined with 266 siRNAs targeting human EG5, as well as its rhesus monkey ortholog (Table 2). An expanded screening set was selected with 328 siRNA targeting human EG5, with no necessity to hit any EG5 mRNA of other species (Table 3).

The sequences for human and a partial rhesus EG5 mRNAs were downloaded from NCBI Nucleotide database and the human sequence was further on used as reference sequence (Human EG5:NM_(—)004523.2, 4908 bp, and Rhesus EG5: XM_(—)001087644.1, 878 bp (only 5′ part of human EG5)

For identification of further rhesus EG5 sequences a mega blast search with the human sequence was conducted at NCBI against rhesus reference genome. The downloaded rhesus sequence and the hit regions in the blast hit were assembled to a rhesus consensus sequence with ˜92% identity to human EG5 over the full-length.

All possible 19mers were extracted from the human mRNA sequence, resulting in the pool of candidate target sites corresponding to 4890 (sense strand) sequences of human-reactive EG5 siRNAs.

Human-rhesus cross-reactivity as prerequisite for in silico selection of siRNAs for an initial screening set out of this candidate pool. To determine rhesus-reactive siRNAs, each candidate siRNA target site was searched for presence in the assembled rhesus sequence. Further, the predicted specificity of the siRNA as criterion for selection of out the pool of human-rhesus cross-reactive siRNAs, manifested by targeting human EG5 mRNA sequences, but not other human mRNAs.

The specificity of an siRNA can be expressed via its potential to target other genes, which are referred to as “off-target genes”.

For predicting the off-target potential of an siRNA, the following assumptions were made:

-   -   1) off-target potential of a strand can be deduced from the         number and distribution of mismatches to an off-target     -   2) the most relevant off-target, that is the gene predicted to         have the highest probability to be silenced due to tolerance of         mismatches, determines the off-target potential of the strand     -   3) positions 2 to 9 (counting 5′ to 3′) of a strand (seed         region) may contribute more to off-target potential than rest of         sequence (that is non-seed and cleavage site region)     -   4) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage         site region) may contribute more to off-target potential than         non-seed region (that is positions 12 to 18, counting 5′ to 3′)     -   5) positions 1 and 19 of each strand are not relevant for         off-target interactions     -   6) off-target potential can be expressed by the off-target score         of the most relevant off-target, calculated based on number and         position of mismatches of the strand to the most homologous         region in the off-target gene considering assumptions 3 to 5     -   7) off-target potential of antisense and sense strand will be         relevant, whereas potential abortion of sense strand activity by         internal modifications introduced is likely

SiRNAs with low off-target potential were defined as preferable and assumed to be more specific.

In order to identify human EG5-specific siRNAs, all other human transcripts, which were all considered potential off-targets, were searched for potential target regions for human-rhesus cross-reactive 19mer sense strand sequences as well as complementary antisense strands. For this, the fastA algorithm was used to determine the most homologues hit region in each sequence of the human RefSeq database, which we assume to represent the comprehensive human transcriptome.

To rank all potential off-targets according to assumptions 3 to 5, and by this identify the most relevant off-target gene and its off-target score, fastA output files were analyzed further by a perl script.

The script extracted the following off-target properties for each 19mer input sequence and each off-target gene to calculate the off-target score:

Number of mismatches in non-seed region

Number of mismatches in seed region

Number of mismatches in cleavage site region

The off-target score was calculated by considering assumptions 3 to 5 as follows:

Off-target score=number of seed mismatches*10

+number of cleavage site mismatches*1.2

+number of non-seed mismatches*1

The most relevant off-target gene for each 19mer sequence was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as representative for the off-target potential of a strand.

For the screening set in Table 2, an off-target score of 3 or more for the antisense strand and 2 or more for the sense strand was chosen as prerequisite for selection of siRNAs, whereas all sequences containing 4 or more consecutive G's (poly-G sequences) were excluded. 266 human-rhesus cross-reactive sequences passing the specificity criterion, were selected based on this cut-off (see Table 2).

For definition of the expanded screening set the cross-reactivity to rhesus was disgarded, re-calculated the predicted specificity based on the newly available human RefSeq database and selected only those 328 non-poly-G siRNAs with off-target score of 2,2 or more for the antisense and sense strand (see Table 3).

For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine: u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.

TABLE 1 TABLE 1A position SEQ sequence of SEQ sense SEQ antisense in human ID total 23mer ID sequence ID sequence duplex access. # NO: target site No: (5′-3′) No: (5′-3′) name 385-407 1244 ACCGAAGUGUUG  1 cGAAGuGuuGu  2 UUGGAcAAAcA AL-DP- UUUGUCCAAUU uuGuccAATsT AcACUUCGTsT 6226 347-369 1245 UAUGGUGUUUGG  3 uGGuGuuuGGA  4 GuAGAUGCUCc AL-DP- AGCAUCUACUA GcAucuAcTsT AAAcACcATsT 6227 1078-1100 1246 AAUCUAAACUAA  5 ucuAAAcuAAc  6 GGAUUCuAGUu AL-DP- CUAGAAUCCUC uAGAAuccTsT AGUUuAGATsT 6228 1067-1089 1247 UCCUUAUCGAGA  7 cuuAucGAGAA  8 AGUUuAGAUUC AL-DP- AUCUAAACUAA ucuAAAcuTsT UCGAuAAGTsT 6229 374-396 1248 GAUUGAUGUUUA  9 uuGAuGuuuAc 10 AcACUUCGGuA AL-DP- CCGAAGUGUUG cGAAGuGuTsT AAcAUcAATsT 6230 205-227 1249 UGGUGAGAUGCA 11 GuGAGAuGcAG 12 uAAAUGGUCUG AL-DP- GACCAUUUAAU AccAuuuATsT cAUCUcACTsT 6231 1176-1198 1250 ACUCUGAGUACA 13 ucuGAGuAcAu 14 AuAUUCcAAUG AL-DP- UUGGAAUAUGC uGGAAuAuTsT uACUcAGATsT 6232 386-408 1251 CCGAAGUGUUGU 15 GAAGuGuuGuu 16 AUUGGAcAAAc AL-DP- UUGUCCAAUUC uGuccAAuTsT AAcACUUCTsT 6233 416-438 1252 AGUUAUUAUGGG 17 uuAuuAuGGGc 18 cAAUuAuAGCC AL-DP- CUAUAAUUGCA uAuAAuuGTsT cAuAAuAATsT 6234 485-507 1253 GGAAGGUGAAAG 19 AAGGuGAAAGG 20 UuAGGUGACCU AL-DP- GUCACCUAAUG ucAccuAATsT UUcACCUUTsT 6235 476-498 1254 UUUUACAAUGGA 21 uuAcAAuGGAA 22 CUUUcACCUUC AL-DP- AGGUGAAAGGU GGuGAAAGTsT cAUUGuAATsT 6236 486-508 1255 GAAGGUGAAAGG 23 AGGuGAAAGGu 24 AUuAGGUGACC AL-DP- UCACCUAAUGA cAccuAAuTsT UUUcACCUTsT 6237 487-509 1256 AAGGUGAAAGGU 25 GGuGAAAGGuc 26 cAUuAGGUGAC AL-DP- CACCUAAUGAA AccuAAuGTsT CUUUcACCTsT 6238 1066-1088 1257 UUCCUUAUCGAG 27 ccuuAucGAGA 28 GUUuAGAUUCU AL-DP- AAUCUAAACUA AucuAAAcTsT CGAuAAGGTsT 6239 1256-1278 1258 AGCUCUUAUUAA 29 cucuuAuuAAG 30 GuAuACUCCUu AL-DP- GGAGUAUACGG GAGuAuAcTsT AAuAAGAGTsT 6240 2329-2351 1259 CAGAGAGAUUCU 31 GAGAGAuucuG 32 CcAAAGcAcAG AL-DP- GUGCUUUGGAG uGcuuuGGTsT AAUCUCUCTsT 6241 1077-1099 1260 GAAUCUAAACUA 33 AucuAAAcuAA 34 GAUUCuAGUuA AL-DP- ACUAGAAUCCU cuAGAAucTsT GUUuAGAUTsT 6242 1244-1266 1261 ACUCACCAAAAA 35 ucAccAAAAAA 36 AuAAGAGCUUU AL-DP- AGCUCUUAUUA GcucuuAuTsT UUUGGUGATsT 6243 637-659 1262 AAGAGCUUUUUG 37 GAGcuuuuuGA 38 uAAGAAGAUcA AL-DP- AUCUUCUUAAU ucuucuuATsT AAAAGCUCTsT 6244 1117-1139 1263 GGCGUACAAGAA 39 cGuAcAAGAAc 40 UuAuAGAUGUU AL-DP- CAUCUAUAAUU AucuAuAATsT CUUGuACGTsT 6245 373-395 1264 AGAUUGAUGUUU 41 AuuGAuGuuuA 42 cACUUCGGuAA AL-DP- ACCGAAGUGUU ccGAAGuGTsT AcAUcAAUTsT 6246 1079-1101 1265 AUCUAAACUAAC 43 cuAAAcuAAcu 44 AGGAUUCuAGU AL-DP- UAGAAUCCUCC AGAAuccuTsT uAGUUuAGTsT 6247 383-405 1266 UUACCGAAGUGU 45 AccGAAGuGuu 46 GGAcAAAcAAc AL-DP- UGUUUGUCCAA GuuuGuccTsT ACUUCGGUTsT 6248 200-222 1267 GGUGGUGGUGAG 47 uGGuGGuGAGA 48 GGUCUGcAUCU AL-DP- AUGCAGACCAU uGcAGAccTsT cACcACcATsT 6249 TABLE 1B single dose SDs 2nd screen @ screen 25 nM [% (among duplex residual quadru- name mRNA] plicates) AL-DP-6226  23%  3% AL-DP-6227  69% 10% AL-DP-6228  33%  2% AL-DP-6229   2%  2% AL-DP-6230  66% 11% AL-DP-6231  17%  1% AL-DP-6232   9%  3% AL-DP-6233  24%  6% AL-DP-6234  91%  2% AL-DP-6235 112%  4% AL-DP-6236  69%  4% AL-DP-6237  42%  2% AL-DP-6238  45%  2% AL-DP-6239   2%  1% AL-DP-6240  48%  2% AL-DP-6241  41%  2% AL-DP-6242   8%  2% AL-DP-6243   7%  1% AL-DP-6244   6%  2% AL-DP-6245  12%  2% AL-DP-6246  28%  3% AL-DP-6247  71%  4% AL-DP-6248   5%  2% AL-DP-6249  28%  3%

TABLE 2 TABLE 2A position SEQ sequence of SEQ sense SEQ antisense in human ID total 19mer ID sequence ID sequence duplex access. # NO: target site NO: (5′-3′) NO: (5′-3′) name 829-847 1268 CAUACUCUAG  49 cAuAcucuAGu  50 UGGGAACGACu AD- UCGUUCCCA cGuucccATsT AGAGuAUGTsT 12072 246-264 1269 AGCGCCCAUU  51 AGcGcccAuuc  52 CuACuAUUGAA AD- CAAUAGUAG AAuAGuAGTsT UGGGCGCUTsT 12073 238-256 1270 GGAAAGCUAG  53 GGAAAGcuAGc  54 GAAUGGGCGCu AD- CGCCCAUUC GcccAuucTsT AGCUUUCCTsT 12074 239-257 1271 GAAAGCUAGC  55 GAAAGcuAGcG  56 UGAAUGGGCGC AD- GCCCAUUCA cccAuucATsT uAGCUUUCTsT 12075 878-896 1272 AGAAACUACG  57 AGAAAcuAcGA  58 UCcAUcAAUCG AD- AUUGAUGGA uuGAuGGATsT uAGUUUCUTsT 12076 1064-1082 1273 UGUUCCUUAU  59 uGuuccuuAuc  60 AGAUUCUCGAu AD- CGAGAAUCU GAGAAucuTsT AAGGAAcATsT 12077 3278-3296 1274 CAGAUUACCU  61 cAGAuuAccuc  62 GGCUCGcAGAG AD- CUGCGAGCC uGcGAGccTsT GuAAUCUGTsT 12078 247-265 1275 GCGCCCAUUC  63 GcGcccAuucA  64 UCuACuAUUGA AD- AAUAGUAGA AuAGuAGATsT AUGGGCGCTsT 12079 434-452 1276 UUGCACUAUC  65 uuGcAcuAucu  66 AuACGcAAAGA AD- UUUGCGUAU uuGcGuAuTsT uAGUGcAATsT 12080 232-250 1277 CAGAGCGGAA  67 cAGAGcGGAAA  68 GCGCuAGCUUU AD- AGCUAGCGC GcuAGcGcTsT CCGCUCUGTsT 12081 1831-1849 1278 AGACCUUAUU  69 AGAccuuAuuu  70 AGAUuACcAAA AD- UGGUAAUCU GGuAAucuTsT uAAGGUCUTsT 12082 1105-1123 1279 AUUCUCUUGG  71 AuucucuuGGA  72 GuACGCCCUCc AD- AGGGCGUAC GGGcGuAcTsT AAGAGAAUTsT 12083 536-554 1280 GGCUGGUAUA  73 GGcuGGuAuAA  74 ACGUGGAAUuA AD- AUUCCACGU uuccAcGuTsT uACcAGCCTsT 12084 236-254 1281 GCGGAAAGCU  75 GcGGAAAGcuA  76 AUGGGCGCuAG AD- AGCGCCCAU GcGcccAuTsT CUUUCCGCTsT 12085 435-453 1282 UGCACUAUCU  77 uGcAcuAucuu  78 cAuACGcAAAG AD- UUGCGUAUG uGcGuAuGTsT AuAGUGcATsT 12086 541-559 1283 GUAUAAUUCC  79 GuAuAAuuccA  80 AGGGuACGUGG AD- ACGUACCCU cGuAcccuTsT AAUuAuACTsT 12087 1076-1094 1284 AGAAUCUAAA  81 AGAAucuAAAc  82 UCuAGUuAGUU AD- CUAACUAGA uAAcuAGATsT uAGAUUCUTsT 12088 1432-1450 1285 AGGAGCUGAA  83 AGGAGcuGAAu  84 GuAACCCuAUU AD- UAGGGUUAC AGGGuuAcTsT cAGCUCCUTsT 12089 1821-1839 1286 GAAGUACAUA  85 GAAGuAcAuAA  86 AuAAGGUCUuA AD- AGACCUUAU GAccuuAuTsT UGuACUUCTsT 12090 2126-2144 1287 GACAGUGGCC  87 GAcAGuGGccG  88 uAUCUuAUCGG AD- GAUAAGAUA AuAAGAuATsT CcACUGUCTsT 12091 2373-2391 1288 AAACCACUUA  89 AAAccAcuuAG  90 GGAcACuACuA AD- GUAGUGUCC uAGuGuccTsT AGUGGUUUTsT 12092 4026-4044 1289 UCCCUAGACU  91 ucccuAGAcuu  92 AAAuAGGGAAG AD- UCCCUAUUU cccuAuuuTsT UCuAGGGATsT 12093 4030-4048 1290 UAGACUUCCC  93 uAGAcuucccu  94 AGCGAAAuAGG AD- UAUUUCGCU AuuucGcuTsT GAAGUCuATsT 12094 144-162 1291 GCGUCGCAGC  95 GcGucGcAGcc  96 ACGAAUUUGGC AD- CAAAUUCGU AAAuucGuTsT UGCGACGCTsT 12095 242-260 1292 AGCUAGCGCC  97 AGcuAGcGccc  98 uAUUGAAUGGG AD- CAUUCAAUA AuucAAuATsT CGCuAGCUTsT 12096 879-897 1293 GAAACUACGA  99 GAAAcuAcGAu 100 CUCcAUcAAUC AD- UUGAUGGAG uGAuGGAGTsT GuAGUUUCTsT 12097 2134-2152 1294 CCGAUAAGAU 101 ccGAuAAGAuA 102 UGAUCUUCuAU AD- AGAAGAUCA GAAGAucATsT CUuAUCGGTsT 12098 245-263 1295 UAGCGCCCAU 103 uAGcGcccAuu 104 uACuAUUGAAU AD- UCAAUAGUA cAAuAGuATsT GGGCGCuATsT 12099 444-462 1296 UUUGCGUAUG 105 uuuGcGuAuGG 106 cAGUUUGGCcA AD- GCCAAACUG ccAAAcuGTsT uACGcAAATsT 12100 550-568 1297 CACGUACCCU 107 cAcGuAcccuu 108 AUUUGAUGAAG AD- UCAUCAAAU cAucAAAuTsT GGuACGUGTsT 12101 442-460 1298 UCUUUGCGUA 109 ucuuuGcGuAu 110 GUUUGGCcAuA AD- UGGCCAAAC GGccAAAcTsT CGcAAAGATsT 12102 386-404 1299 CCGAAGUGUU 111 ccGAAGuGuuG 112 UGGAcAAAcAA AD- GUUUGUCCA uuuGuccATsT cACUUCGGTsT 12103 233-251 1300 AGAGCGGAAA 113 AGAGcGGAAAG 114 GGCGCuAGCUU AD- GCUAGCGCC cuAGcGccTsT UCCGCUCUTsT 12104 243-261 1301 GCUAGCGCCC 115 GcuAGcGcccA 116 CuAUUGAAUGG AD- AUUCAAUAG uucAAuAGTsT GCGCuAGCTsT 12105 286-304 1302 AAGUUAGUGU 117 AAGuuAGuGuA 118 CcAGUUCGuAc AD- ACGAACUGG cGAAcuGGTsT ACuAACUUTsT 12106 294-312 1303 GUACGAACUG 119 GuAcGAAcuGG 120 CcAAUCCUCcA AD- GAGGAUUGG AGGAuuGGTsT GUUCGuACTsT 12107 296-314 1304 ACGAACUGGA 121 AcGAAcuGGAG 122 AGCcAAUCCUC AD- GGAUUGGCU GAuuGGcuTsT cAGUUCGUTsT 12108 373-391 1305 AGAUUGAUGU 123 AGAuuGAuGuu 124 CUUCGGuAAAc AD- UUACCGAAG uAccGAAGTsT AUcAAUCUTsT 12109 422-440 1306 UAUGGGCUAU 125 uAuGGGcuAuA 126 AGUGcAAUuAu AD- AAUUGCACU AuuGcAcuTsT AGCCcAuATsT 12110 441-459 1307 AUCUUUGCGU 127 AucuuuGcGuA 128 UUUGGCcAuAC AD- AUGGCCAAA uGGccAAATsT GcAAAGAUTsT 12111 832-850 1308 ACUCUAGUCG 129 AcucuAGucGu 130 GAGUGGGAACG AD- UUCCCACUC ucccAcucTsT ACuAGAGUTsT 12112 881-899 1309 AACUACGAUU 131 AAcuAcGAuuG 132 UUCUCcAUcAA AD- GAUGGAGAA AuGGAGAATsT UCGuAGUUTsT 12113 975-993 1310 GAUAAGAGAG 133 GAuAAGAGAGc 134 CUUCCCGAGCU AD- CUCGGGAAG ucGGGAAGTsT CUCUuAUCTsT 12114 1073-1091 1311 UCGAGAAUCU 135 ucGAGAAucuA 136 AGUuAGUUuAG AD- AAACUAACU AAcuAAcuTsT AUUCUCGATsT 12115 1084-1102 1312 AACUAACUAG 137 AAcuAAcuAGA 138 UGGAGGAUUCu AD- AAUCCUCCA AuccuccATsT AGUuAGUUTsT 12116 1691-1709 1313 GGAUCGUAAG 139 GGAucGuAAGA 140 AACUGCCUUCU AD- AAGGCAGUU AGGcAGuuTsT uACGAUCCTsT 12117 1693-1711 1314 AUCGUAAGAA 141 AucGuAAGAAG 142 UcAACUGCCUU AD- GGCAGUUGA GcAGuuGATsT CUuACGAUTsT 12118 1702-1720 1315 AGGCAGUUGA 143 AGGcAGuuGAc 144 UUGUGUUGGUc AD- CCAACACAA cAAcAcAATsT AACUGCCUTsT 12119 2131-2149 1316 UGGCCGAUAA 145 uGGccGAuAAG 146 UCUUCuAUCUu AD- GAUAGAAGA AuAGAAGATsT AUCGGCcATsT 12120 2412-2430 1317 UCUAAGGAUA 147 ucuAAGGAuAu 148 UGUUGACuAuA AD- UAGUCAACA AGucAAcATsT UCCUuAGATsT 12121 2859-2877 1318 ACUAAGCUUA 149 AcuAAGcuuAA 150 GAAAGcAAUuA AD- AUUGCUUUC uuGcuuucTsT AGCUuAGUTsT 12122 3294-3312 1319 GCCCAGAUCA 151 GcccAGAucAA 152 AUuAAAGGUUG AD- ACCUUUAAU ccuuuAAuTsT AUCUGGGCTsT 12123 223-241 1320 UUAAUUUGGC 153 uuAAuuuGGcA 154 UUCCGCUCUGC AD- AGAGCGGAA GAGcGGAATsT cAAAUuAATsT 12124 1070-1088 1321 UUAUCGAGAA 155 uuAucGAGAAu 156 uAGUUuAGAUU AD- UCUAAACUA cuAAAcuATsT CUCGAuAATsT 12125 244-262 1322 CUAGCGCCCA 157 cuAGcGcccAu 158 ACuAUUGAAUG AD- UUCAAUAGU ucAAuAGuTsT GGCGCuAGTsT 12126 257-275 1323 AAUAGUAGAA 159 AAuAGuAGAAu 160 AGGAUcAcAUU AD- UGUGAUCCU GuGAuccuTsT CuACuAUUTsT 12127 277-295 1324 UACGAAAAGA 161 uAcGAAAAGAA 162 AcACuAACUUC AD- AGUUAGUGU GuuAGuGuTsT UUUUCGuATsT 12128 284-302 1325 AGAAGUUAGU 163 AGAAGuuAGuG 164 AGUUCGuAcAC AD- GUACGAACU uAcGAAcuTsT uAACUUCUTsT 12129 366-384 1326 ACUAAACAGA 165 AcuAAAcAGAu 166 AAAcAUcAAUC AD- UUGAUGUUU uGAuGuuuTsT UGUUuAGUTsT 12130 443-461 1327 CUUUGCGUAU 167 cuuuGcGuAuG 168 AGUUUGGCcAu AD- GGCCAAACU GccAAAcuTsT ACGcAAAGTsT 12131 504-522 1328 AAUGAAGAGU 169 AAuGAAGAGuA 170 CCcAGGuAuAC AD- AUACCUGGG uAccuGGGTsT UCUUcAUUTsT 12132 543-561 1329 AUAAUUCCAC 171 AuAAuuccAcG 172 GAAGGGuACGU AD- GUACCCUUC uAcccuucTsT GGAAUuAUTsT 12133 551-569 1330 ACGUACCCUU 173 AcGuAcccuuc 174 AAUUUGAUGAA AD- CAUCAAAUU AucAAAuuTsT GGGuACGUTsT 12134 552-570 1331 CGUACCCUUC 175 cGuAcccuucA 176 AAAUUUGAUGA AD- AUCAAAUUU ucAAAuuuTsT AGGGuACGTsT 12135 553-571 1332 GUACCCUUCA 177 GuAcccuucAu 178 AAAAUUUGAUG AD- UCAAAUUUU cAAAuuuuTsT AAGGGuACTsT 12136 577-595 1333 AACUUACUGA 179 AAcuuAcuGAu 180 GuACcAUuAUc AD- UAAUGGUAC AAuGGuAcTsT AGuAAGUUTsT 12137 602-620 1334 UUCAGUCAAA 181 uucAGucAAAG 182 cAGAGAcACUU AD- GUGUCUCUG uGucucuGTsT UGACUGAATsT 12138 652-670 1335 UUCUUAAUCC 183 uucuuAAuccA 184 UcAGAUGAUGG AD- AUCAUCUGA ucAucuGATsT AUuAAGAATsT 12139 747-765 1336 ACAGUACACA 185 AcAGuAcAcAA 186 cAUCCUUGUUG AD- ACAAGGAUG cAAGGAuGTsT UGuACUGUTsT 12140 877-895 1337 AAGAAACUAC 187 AAGAAAcuAcG 188 CcAUcAAUCGu AD- GAUUGAUGG AuuGAuGGTsT AGUUUCUUTsT 12141 880-898 1338 AAACUACGAU 189 AAAcuAcGAuu 190 UCUCcAUcAAU AD- UGAUGGAGA GAuGGAGATsT CGuAGUUUTsT 12142 965-983 1339 UGGAGCUGUU 191 uGGAGcuGuuG 192 UCUCUuAUcAA AD- GAUAAGAGA AuAAGAGATsT cAGCUCcATsT 12143 1086-1104 1340 CUAACUAGAA 193 cuAAcuAGAAu 194 CCUGGAGGAUU AD- UCCUCCAGG ccuccAGGTsT CuAGUuAGTsT 12144 1191-1209 1341 GAAUAUGCUC 195 GAAuAuGcucA 196 UUGCUCuAUGA AD- AUAGAGCAA uAGAGcAATsT GcAuAUUCTsT 12145 1195-1213 1342 AUGCUCAUAG 197 AuGcucAuAGA 198 UUCUUUGCUCu AD- AGCAAAGAA GcAAAGAATsT AUGAGcAUTsT 12146 1412-1430 1343 AAAAAUUGGU 199 AAAAAuuGGuG 200 CUcAAcAGcAC AD- GCUGUUGAG cuGuuGAGTsT cAAUUUUUTsT 12147 1431-1449 1344 GAGGAGCUGA 201 GAGGAGcuGAA 202 uAACCCuAUUc AD- AUAGGGUUA uAGGGuuATsT AGCUCCUCTsT 12148 1433-1451 1345 GGAGCUGAAU 203 GGAGcuGAAuA 204 UGuAACCCuAU AD- AGGGUUACA GGGuuAcATsT UcAGCUCCTsT 12149 1434-1452 1346 GAGCUGAAUA 205 GAGcuGAAuAG 206 CUGuAACCCuA AD- GGGUUACAG GGuuAcAGTsT UUcAGCUCTsT 12150 1435-1453 1347 AGCUGAAUAG 207 AGcuGAAuAGG 208 UCUGuAACCCu AD- GGUUACAGA GuuAcAGATsT AUUcAGCUTsT 12151 1436-1454 1348 GCUGAAUAGG 209 GcuGAAuAGGG 210 CUCUGuAACCC AD- GUUACAGAG uuAcAGAGTsT uAUUcAGCTsT 12152 1684-1702 1349 CCAAACUGGA 211 ccAAAcuGGAu 212 UUCUuACGAUC AD- UCGUAAGAA cGuAAGAATsT cAGUUUGGTsT 12153 1692-1710 1350 GAUCGUAAGA 213 GAucGuAAGAA 214 cAACUGCCUUC AD- AGGCAGUUG GGcAGuuGTsT UuACGAUCTsT 12154 1833-1851 1351 ACCUUAUUUG 215 AccuuAuuuGG 216 GcAGAUuACcA AD- GUAAUCUGC uAAucuGcTsT AAuAAGGUTsT 12155 1872-1890 1352 UUAGAUACCA 217 uuAGAuAccAu 218 CUGuAGuAAUG AD- UUACUACAG uAcuAcAGTsT GuAUCuAATsT 12156 1876-1894 1353 AUACCAUUAC 219 AuAccAuuAcu 220 GCuACUGuAGu AD- UACAGUAGC AcAGuAGcTsT AAUGGuAUTsT 12157 1883-1901 1354 UACUACAGUA 221 uAcuAcAGuAG 222 UCcAAGUGCuA AD- GCACUUGGA cAcuuGGATsT CUGuAGuATsT 12158 1987-2005 1355 AAAGUAAAAC 223 AAAGuAAAAcu 224 UGuAGuAcAGU AD- UGUACUACA GuAcuAcATsT UUuACUUUTsT 12159 2022-2040 1356 CUCAAGACUG 225 cucAAGAcuGA 226 UuAGAAGAUcA AD- AUCUUCUAA ucuucuAATsT GUCUUGAGTsT 12160 2124-2142 1357 UUGACAGUGG 227 uuGAcAGuGGc 228 UCUuAUCGGCc AD- CCGAUAAGA cGAuAAGATsT ACUGUcAATsT 12161 2125-2143 1358 UGACAGUGGC 229 uGAcAGuGGcc 230 AUCUuAUCGGC AD- CGAUAAGAU GAuAAGAuTsT cACUGUcATsT 12162 2246-2264 1359 GCAAUGUGGA 231 GcAAuGuGGAA 232 AGUuAGGUUUC AD- AACCUAACU AccuAAcuTsT cAcAUUGCTsT 12163 2376-2394 1360 CCACUUAGUA 233 ccAcuuAGuAG 234 CCUGGAcACuA AD- GUGUCCAGG uGuccAGGTsT CuAAGUGGTsT 12164 2504-2522 1361 AGAAGGUACA 235 AGAAGGuAcAA 236 AACcAAUUUUG AD- AAAUUGGUU AAuuGGuuTsT uACCUUCUTsT 12165 2852-2870 1362 UGGUUUGACU 237 uGGuuuGAcuA 238 AUuAAGCUuAG AD- AAGCUUAAU AGcuuAAuTsT UcAAACcATsT 12166 2853-2871 1363 GGUUUGACUA 239 GGuuuGAcuAA 240 AAUuAAGCUuA AD- AGCUUAAUU GcuuAAuuTsT GUcAAACCTsT 12167 3110-3128 1364 UCUAAGUCAA 241 ucuAAGucAAG 242 AGAUGGCUCUU AD- GAGCCAUCU AGccAucuTsT GACUuAGATsT 12168 3764-3782 1365 UCAUCCCUAU 243 ucAucccuAuA 244 AAGUGAACuAu AD- AGUUCACUU GuucAcuuTsT AGGGAUGATsT 12169 3765-3783 1366 CAUCCCUAUA 245 cAucccuAuAG 246 AAAGUGAACuA AD- GUUCACUUU uucAcuuuTsT uAGGGAUGTsT 12170 4027-4045 1367 CCCUAGACUU 247 cccuAGAcuuc 248 GAAAuAGGGAA AD- CCCUAUUUC ccuAuuucTsT GUCuAGGGTsT 12171 4031-4049 1368 AGACUUCCCU 249 AGAcuucccuA 250 AAGCGAAAuAG AD- AUUUCGCUU uuucGcuuTsT GGAAGUCUTsT 12172 4082-4100 1369 UCACCAAACC 251 ucAccAAAccA 252 UCuAcAAAUGG AD- AUUUGUAGA uuuGuAGATsT UUUGGUGATsT 12173 4272-4290 1370 UCCUUUAAGA 253 uccuuuAAGAG 254 AGUuAGGCCUC AD- GGCCUAACU GccuAAcuTsT UuAAAGGATsT 12174 4275-4293 1371 UUUAAGAGGC 255 uuuAAGAGGcc 256 AUGAGUuAGGC AD- CUAACUCAU uAAcucAuTsT CUCUuAAATsT 12175 4276-4294 1372 UUAAGAGGCC 257 uuAAGAGGccu 258 AAUGAGUuAGG AD- UAACUCAUU AAcucAuuTsT CCUCUuAATsT 12176 4282-4300 1373 GGCCUAACUC 259 GGccuAAcucA 260 AGGGUGAAUGA AD- AUUCACCCU uucAcccuTsT GUuAGGCCTsT 12177 4571-4589 1374 UGGUAUUUUU 261 uGGuAuuuuuG 262 UGCcAGAUcAA AD- GAUCUGGCA AucuGGcATsT AAAuACcATsT 12178 4677-4695 1375 AGUUUAGUGU 263 AGuuuAGuGuG 264 AAACUUuAcAc AD- GUAAAGUUU uAAAGuuuTsT ACuAAACUTsT 12179 152-170 1376 GCCAAAUUCG 265 GccAAAuucGu 266 CUUCGcAGACG AD- UCUGCGAAG cuGcGAAGTsT AAUUUGGCTsT 12180 156-174 1377 AAUUCGUCUG 267 AAuucGucuGc 268 UCUUCUUCGcA AD- CGAAGAAGA GAAGAAGATsT GACGAAUUTsT 12181 491-509 1378 UGAAAGGUCA 269 uGAAAGGucAc 270 UUcAUuAGGUG AD- CCUAAUGAA cuAAuGAATsT ACCUUUcATsT 12182 215-233 1379 CAGACCAUUU 271 cAGAccAuuuA 272 UGCcAAAUuAA AD- AAUUUGGCA AuuuGGcATsT AUGGUCUGTsT 12183 216-234 1380 AGACCAUUUA 273 AGAccAuuuAA 274 CUGCcAAAUuA AD- AUUUGGCAG uuuGGcAGTsT AAUGGUCUTsT 12184 416-434 1381 AGUUAUUAUG 275 AGuuAuuAuGG 276 AUuAuAGCCcA AD- GGCUAUAAU GcuAuAAuTsT uAAuAACUTsT 12185 537-555 1382 GCUGGUAUAA 277 GcuGGuAuAAu 278 uACGUGGAAUu AD- UUCCACGUA uccAcGuATsT AuACcAGCTsT 12186 221-239 1383 AUUUAAUUUG 279 AuuuAAuuuGG 280 CCGCUCUGCcA AD- GCAGAGCGG cAGAGcGGTsT AAUuAAAUTsT 12187 222-240 1384 UUUAAUUUGG 281 uuuAAuuuGGc 282 UCCGCUCUGCc AD- CAGAGCGGA AGAGcGGATsT AAAUuAAATsT 12188 227-245 1385 UUUGGCAGAG 283 uuuGGcAGAGc 284 AGCUUUCCGCU AD- CGGAAAGCU GGAAAGcuTsT CUGCcAAATsT 12189 476-494 1386 UUUUACAAUG 285 uuuuAcAAuGG 286 UUcACCUUCcA AD- GAAGGUGAA AAGGuGAATsT UUGuAAAATsT 12190 482-500 1387 AAUGGAAGGU 287 AAuGGAAGGuG 288 UGACCUUUcAC AD- GAAAGGUCA AAAGGucATsT CUUCcAUUTsT 12191 208-226 1388 UGAGAUGCAG 289 uGAGAuGcAGA 290 UuAAAUGGUCU AD- ACCAUUUAA ccAuuuAATsT GcAUCUcATsT 12192 147-165 1389 UCGCAGCCAA 291 ucGcAGccAAA 292 cAGACGAAUUU AD- AUUCGUCUG uucGucuGTsT GGCUGCGATsT 12193 426-444 1390 GGCUAUAAUU 293 GGcuAuAAuuG 294 AGAuAGUGcAA AD- GCACUAUCU cAcuAucuTsT UuAuAGCCTsT 12194 2123-2141 1391 AUUGACAGUG 295 AuuGAcAGuGG 296 CUuAUCGGCcA AD- GCCGAUAAG ccGAuAAGTsT CUGUcAAUTsT 12195 4029-4047 1392 CUAGACUUCC 297 cuAGAcuuccc 298 GCGAAAuAGGG AD- CUAUUUCGC uAuuucGcTsT AAGUCuAGTsT 12196 438-456 1393 ACUAUCUUUG 299 AcuAucuuuGc 300 GGCcAuACGcA AD- CGUAUGGCC GuAuGGccTsT AAGAuAGUTsT 12197 830-848 1394 AUACUCUAGU 301 AuAcucuAGuc 302 GUGGGAACGAC AD- CGUUCCCAC GuucccAcTsT uAGAGuAUTsT 12198 876-894 1395 AAAGAAACUA 303 AAAGAAAcuAc 304 cAUcAAUCGuA AD- CGAUUGAUG GAuuGAuGTsT GUUUCUUUTsT 12199 115-133 1396 GCCUUGAUUU 305 GccuuGAuuuu 306 CCCGCcAAAAA AD- UUUGGCGGG uuGGcGGGTsT AUcAAGGCTsT 12200 248-266 1397 CGCCCAUUCA 307 cGcccAuucAA 308 UUCuACuAUUG AD- AUAGUAGAA uAGuAGAATsT AAUGGGCGTsT 12201 1834-1852 1398 CCUUAUUUGG 309 ccuuAuuuGGu 310 AGcAGAUuACc AD- UAAUCUGCU AAucuGcuTsT AAAuAAGGTsT 12202 3050-3068 1399 AGAGACAAUU 311 AGAGAcAAuuc 312 cAcAUCCGGAA AD- CCGGAUGUG cGGAuGuGTsT UUGUCUCUTsT 12203 4705-4723 1400 UGACUUUGAU 313 uGAcuuuGAuA 314 AAUUuAGCuAU AD- AGCUAAAUU GcuAAAuuTsT cAAAGUcATsT 12204 229-247 1401 UGGCAGAGCG 315 uGGcAGAGcGG 316 CuAGCUUUCCG AD- GAAAGCUAG AAAGcuAGTsT CUCUGCcATsT 12205 234-252 1402 GAGCGGAAAG 317 GAGcGGAAAGc 318 GGGCGCuAGCU AD- CUAGCGCCC uAGcGcccTsT UUCCGCUCTsT 12206 282-300 1403 AAAGAAGUUA 319 AAAGAAGuuAG 320 UUCGuAcACuA AD- GUGUACGAA uGuAcGAATsT ACUUCUUUTsT 12207 433-451 1404 AUUGCACUAU 321 AuuGcAcuAuc 322 uACGcAAAGAu AD- CUUUGCGUA uuuGcGuATsT AGUGcAAUTsT 12208 540-558 1405 GGUAUAAUUC 323 GGuAuAAuucc 324 GGGuACGUGGA AD- CACGUACCC AcGuAcccTsT AUuAuACCTsT 12209 831-849 1406 UACUCUAGUC 325 uAcucuAGucG 326 AGUGGGAACGA AD- GUUCCCACU uucccAcuTsT CuAGAGuATsT 12210 872-890 1407 UAUGAAAGAA 327 uAuGAAAGAAA 328 AAUCGuAGUUU AD- ACUACGAUU cuAcGAuuTsT CUUUcAuATsT 12211 1815-1833 1408 AUGCUAGAAG 329 AuGcuAGAAGu 330 UCUuAUGuACU AD- UACAUAAGA AcAuAAGATsT UCuAGcAUTsT 12212 1822-1840 1409 AAGUACAUAA 331 AAGuAcAuAAG 332 AAuAAGGUCUu AD- GACCUUAUU AccuuAuuTsT AUGuACUUTsT 12213 3002-3020 1410 ACAGCCUGAG 333 AcAGccuGAGc 334 cAUuAAcAGCU AD- CUGUUAAUG uGuuAAuGTsT cAGGCUGUTsT 12214 3045-3063 1411 AAAGAAGAGA 335 AAAGAAGAGAc 336 CCGGAAUUGUC AD- CAAUUCCGG AAuuccGGTsT UCUUCUUUTsT 12215 3224-3242 1412 CACACUGGAG 337 cAcAcuGGAGA 338 UUuAGACCUCU AD- AGGUCUAAA GGucuAAATsT CcAGUGUGTsT 12216 3226-3244 1413 CACUGGAGAG 339 cAcuGGAGAGG 340 ACUUuAGACCU AD- GUCUAAAGU ucuAAAGuTsT CUCcAGUGTsT 12217 3227-3245 1414 ACUGGAGAGG 341 AcuGGAGAGGu 342 cACUUuAGACC AD- UCUAAAGUG cuAAAGuGTsT UCUCcAGUTsT 12218 145-163 1415 CGUCGCAGCC 343 cGucGcAGccA 344 GACGAAUUUGG AD- AAAUUCGUC AAuucGucTsT CUGCGACGTsT 12219 1700-1718 1416 GAAGGCAGUU 345 GAAGGcAGuuG 346 GUGUUGGUcAA AD- GACCAACAC AccAAcAcTsT CUGCCUUCTsT 12220 4291-4309 1417 CAUUCACCCU 347 cAuucAcccuG 348 AACUCUGUcAG AD- GACAGAGUU AcAGAGuuTsT GGUGAAUGTsT 12221 4278-4296 1418 AAGAGGCCUA 349 AAGAGGccuAA 350 UGAAUGAGUuA AD- ACUCAUUCA cucAuucATsT GGCCUCUUTsT 12222 3051-3069 1419 GAGACAAUUC 351 GAGAcAAuucc 352 CcAcAUCCGGA AD- CGGAUGUGG GGAuGuGGTsT AUUGUCUCTsT 12223 3058-3076 1420 UUCCGGAUGU 353 uuccGGAuGuG 354 UCuAcAUCcAc AD- GGAUGUAGA GAuGuAGATsT AUCCGGAATsT 12224 241-259 1421 AAGCUAGCGC 355 AAGcuAGcGcc 356 AUUGAAUGGGC AD- CCAUUCAAU cAuucAAuTsT GCuAGCUUTsT 12225 285-303 1422 GAAGUUAGUG 357 GAAGuuAGuGu 358 cAGUUCGuAcA AD- UACGAACUG AcGAAcuGTsT CuAACUUCTsT 12226 542-560 1423 UAUAAUUCCA 359 uAuAAuuccAc 360 AAGGGuACGUG AD- CGUACCCUU GuAcccuuTsT GAAUuAuATsT 12227 2127-2145 1424 ACAGUGGCCG 361 AcAGuGGccGA 362 CuAUCUuAUCG AD- AUAAGAUAG uAAGAuAGTsT GCcACUGUTsT 12228 3760-3778 1425 UCUGUCAUCC 363 ucuGucAuccc 364 GAACuAuAGGG AD- CUAUAGUUC uAuAGuucTsT AUGAcAGATsT 12229 3993-4011 1426 UUCUUGCUAU 365 uucuuGcuAuG 366 AcAcAAGUcAu AD- GACUUGUGU AcuuGuGuTsT AGcAAGAATsT 12230 1696-1714 1427 GUAAGAAGGC 367 GuAAGAAGGcA 368 UGGUcAACUGC AD- AGUUGACCA GuuGAccATsT CUUCUuACTsT 12231 2122-2140 1428 CAUUGACAGU 369 cAuuGAcAGuG 370 UuAUCGGCcAC AD- GGCCGAUAA GccGAuAATsT UGUcAAUGTsT 12232 2371-2389 1429 AGAAACCACU 371 AGAAAccAcuu 372 AcACuACuAAG AD- UAGUAGUGU AGuAGuGuTsT UGGUUUCUTsT 12233 3143-3161 1430 GGAUUGUUCA 373 GGAuuGuucAu 374 GCcAAUUGAUG AD- UCAAUUGGC cAAuuGGcTsT AAcAAUCCTsT 12234 4277-4295 1431 UAAGAGGCCU 375 uAAGAGGccuA 376 GAAUGAGUuAG AD- AACUCAUUC AcucAuucTsT GCCUCUuATsT 12235 287-305 1432 AGUUAGUGUA 377 AGuuAGuGuAc 378 UCcAGUUCGuA AD- CGAACUGGA GAAcuGGATsT cACuAACUTsT 12236 1823-1841 1433 AGUACAUAAG 379 AGuAcAuAAGA 380 AAAuAAGGUCU AD- ACCUUAUUU ccuuAuuuTsT uAUGuACUTsT 12237 3379-3397 1434 UGAGCCUUGU 381 uGAGccuuGuG 382 AAUCuAuAcAc AD- GUAUAGAUU uAuAGAuuTsT AAGGCUcATsT 12238 4273-4291 1435 CCUUUAAGAG 383 ccuuuAAGAGG 384 GAGUuAGGCCU AD- GCCUAACUC ccuAAcucTsT CUuAAAGGTsT 12239 2375-2393 1436 ACCACUUAGU 385 AccAcuuAGuA 386 CUGGAcACuAC AD- AGUGUCCAG GuGuccAGTsT uAAGUGGUTsT 12240 4439-4457 1437 GAAACUUCCA 387 GAAAcuuccAA 388 AGAcAuAAUUG AD- AUUAUGUCU uuAuGucuTsT GAAGUUUCTsT 12241 827-845 1438 UGCAUACUCU 389 uGcAuAcucuA 390 GGAACGACuAG AD- AGUCGUUCC GucGuuccTsT AGuAUGcATsT 12242 1699-1717 1439 AGAAGGCAGU 391 AGAAGGcAGuu 392 UGUUGGUcAAC AD- UGACCAACA GAccAAcATsT UGCCUUCUTsT 12243 1824-1842 1440 GUACAUAAGA 393 GuAcAuAAGAc 394 cAAAuAAGGUC AD- CCUUAUUUG cuuAuuuGTsT UuAUGuACTsT 12244 429-447 1441 UAUAAUUGCA 395 uAuAAuuGcAc 396 cAAAGAuAGUG AD- CUAUCUUUG uAucuuuGTsT cAAUuAuATsT 12245 856-874 1442 UCUCUGUUAC 397 ucucuGuuAcA 398 AuAUGuAUUGu AD- AAUACAUAU AuAcAuAuTsT AAcAGAGATsT 12246 1194-1212 1443 UAUGCUCAUA 399 uAuGcucAuAG 400 UCUUUGCUCuA AD- GAGCAAAGA AGcAAAGATsT UGAGcAuATsT 12247 392-410 1444 UGUUGUUUGU 401 uGuuGuuuGuc 402 cAGAAUUGGAc AD- CCAAUUCUG cAAuucuGTsT AAAcAAcATsT 12248 1085-1103 1445 ACUAACUAGA 403 AcuAAcuAGAA 404 CUGGAGGAUUC AD- AUCCUCCAG uccuccAGTsT uAGUuAGUTsT 12249 2069-2087 1446 UGUGGUGUCU 405 uGuGGuGucuA 406 UUUcAGuAuAG AD- AUACUGAAA uAcuGAAATsT AcACcAcATsT 12250 4341-4359 1447 UAUUAUGGGA 407 uAuuAuGGGAG 408 UGGGUGGUCUC AD- GACCACCCA AccAcccATsT CcAuAAuATsT 12251 759-777 1448 AAGGAUGAAG 409 AAGGAuGAAGu 410 UUUGAuAGACU AD- UCUAUCAAA cuAucAAATsT UcAUCCUUTsT 12252 973-991 1449 UUGAUAAGAG 411 uuGAuAAGAGA 412 UCCCGAGCUCU AD- AGCUCGGGA GcucGGGATsT CUuAUcAATsT 12253 1063-1081 1450 AUGUUCCUUA 413 AuGuuccuuAu 414 GAUUCUCGAuA AD- UCGAGAAUC cGAGAAucTsT AGGAAcAUTsT 12254 1190-1208 1451 GGAAUAUGCU 415 GGAAuAuGcuc 416 UGCUCuAUGAG AD- CAUAGAGCA AuAGAGcATsT cAuAUUCCTsT 12255 1679-1697 1452 CCAUUCCAAA 417 ccAuuccAAAc 418 ACGAUCcAGUU AD- CUGGAUCGU uGGAucGuTsT UGGAAUGGTsT 12256 1703-1721 1453 GGCAGUUGAC 419 GGcAGuuGAcc 420 AUUGUGUUGGU AD- CAACACAAU AAcAcAAuTsT cAACUGCCTsT 12257 1814-1832 1454 CAUGCUAGAA 421 cAuGcuAGAAG 422 CUuAUGuACUU AD- GUACAUAAG uAcAuAAGTsT CuAGcAUGTsT 12258 1818-1836 1455 CUAGAAGUAC 423 cuAGAAGuAcA 424 AGGUCUuAUGu AD- AUAAGACCU uAAGAccuTsT ACUUCuAGTsT 12259 1897-1915 1456 UUGGAUCUCU 425 uuGGAucucuc 426 AuAGAUGUGAG AD- CACAUCUAU AcAucuAuTsT AGAUCcAATsT 12260 2066-2084 1457 AACUGUGGUG 427 AAcuGuGGuGu 428 cAGuAuAGAcA AD- UCUAUACUG cuAuAcuGTsT CcAcAGUUTsT 12261 2121-2139 1458 UCAUUGACAG 429 ucAuuGAcAGu 430 uAUCGGCcACU AD- UGGCCGAUA GGccGAuATsT GUcAAUGATsT 12262 2280-2298 1459 AUAAAGCAGA 431 AuAAAGcAGAc 432 GGGAAUGGGUC AD- CCCAUUCCC ccAuucccTsT UGCUUuAUTsT 12263 2369-2387 1460 ACAGAAACCA 433 AcAGAAAccAc 434 ACuACuAAGUG AD- CUUAGUAGU uuAGuAGuTsT GUUUCUGUTsT 12264 2372-2390 1461 GAAACCACUU 435 GAAAccAcuuA 436 GAcACuACuAA AD- AGUAGUGUC GuAGuGucTsT GUGGUUUCTsT 12265 2409-2427 1462 AAAUCUAAGG 437 AAAucuAAGGA 438 UGACuAuAUCC AD- AUAUAGUCA uAuAGucATsT UuAGAUUUTsT 12266 2933-2951 1463 UUAUUUAUAC 439 uuAuuuAuAcc 440 UGUUGAUGGGu AD- CCAUCAACA cAucAAcATsT AuAAAuAATsT 12267 3211-3229 1464 ACAGAGGCAU 441 AcAGAGGcAuu 442 AGUGUGUuAAU AD- UAACACACU AAcAcAcuTsT GCCUCUGUTsT 12268 3223-3241 1465 ACACACUGGA 443 AcAcAcuGGAG 444 UuAGACCUCUC AD- GAGGUCUAA AGGucuAATsT cAGUGUGUTsT 12269 3225-3243 1466 ACACUGGAGA 445 AcAcuGGAGAG 446 CUUuAGACCUC AD- GGUCUAAAG GucuAAAGTsT UCcAGUGUTsT 12270 3291-3309 1467 CGAGCCCAGA 447 cGAGcccAGAu 448 AAAGGUUGAUC AD- UCAACCUUU cAAccuuuTsT UGGGCUCGTsT 12271 4036-4054 1468 UCCCUAUUUC 449 ucccuAuuucG 450 GGAGAAAGCGA AD- GCUUUCUCC cuuucuccTsT AAuAGGGATsT 12272 4180-4198 1469 UCUAAAAUCA 451 ucuAAAAucAc 452 UGUUGAcAGUG AD- CUGUCAACA uGucAAcATsT AUUUuAGATsT 12273 151-169 1470 AGCCAAAUUC 453 AGccAAAuucG 454 UUCGcAGACGA AD- GUCUGCGAA ucuGcGAATsT AUUUGGCUTsT 12274 250-268 1471 CCCAUUCAAU 455 cccAuucAAuA 456 cAUUCuACuAU AD- AGUAGAAUG GuAGAAuGTsT UGAAUGGGTsT 12275 821-839 1472 GAUGAAUGCA 457 GAuGAAuGcAu 458 ACuAGAGuAUG AD- UACUCUAGU AcucuAGuTsT cAUUcAUCTsT 12276 1060-1078 1473 CUCAUGUUCC 459 cucAuGuuccu 460 UCUCGAuAAGG AD- UUAUCGAGA uAucGAGATsT AAcAUGAGTsT 12277 1075-1093 1474 GAGAAUCUAA 461 GAGAAucuAAA 462 CuAGUuAGUUu AD- ACUAACUAG cuAAcuAGTsT AGAUUCUCTsT 12278 1819-1837 1475 UAGAAGUACA 463 uAGAAGuAcAu 464 AAGGUCUuAUG AD- UAAGACCUU AAGAccuuTsT uACUUCuATsT 12279 3003-3021 1476 CAGCCUGAGC 465 cAGccuGAGcu 466 UcAUuAAcAGC AD- UGUUAAUGA GuuAAuGATsT UcAGGCUGTsT 12280 3046-3064 1477 AAGAAGAGAC 467 AAGAAGAGAcA 468 UCCGGAAUUGU AD- AAUUCCGGA AuuccGGATsT CUCUUCUUTsT 12281 3134-3152 1478 UGCUGGUGUG 469 uGcuGGuGuGG 470 UGAAcAAUCcA AD- GAUUGUUCA AuuGuucATsT cACcAGcATsT 12282 155-173 1479 AAAUUCGUCU 471 AAAuucGucuG 472 CUUCUUCGcAG AD- GCGAAGAAG cGAAGAAGTsT ACGAAUUUTsT 12283 4596-4614 1480 UUUCUGGAAG 473 uuucuGGAAGu 474 AcAUCUcAACU AD- UUGAGAUGU uGAGAuGuTsT UCcAGAAATsT 12284 365-383 1481 UACUAAACAG 475 uAcuAAAcAGA 476 AAcAUcAAUCU AD- AUUGAUGUU uuGAuGuuTsT GUUuAGuATsT 12285 374-392 1482 GAUUGAUGUU 477 GAuuGAuGuuu 478 ACUUCGGuAAA AD- UACCGAAGU AccGAAGuTsT cAUcAAUCTsT 12286 436-454 1483 GCACUAUCUU 479 GcAcuAucuuu 480 CcAuACGcAAA AD- UGCGUAUGG GcGuAuGGTsT GAuAGUGCTsT 12287 539-557 1484 UGGUAUAAUU 481 uGGuAuAAuuc 482 GGuACGUGGAA AD- CCACGUACC cAcGuAccTsT UuAuACcATsT 12288 1629-1647 1485 AGCAAGCUGC 483 AGcAAGcuGcu 484 CUGUGUuAAGc AD- UUAACACAG uAAcAcAGTsT AGCUUGCUTsT 12289 2370-2388 1486 CAGAAACCAC 485 cAGAAAccAcu 486 cACuACuAAGU AD- UUAGUAGUG uAGuAGuGTsT GGUUUCUGTsT 12290 2676-2694 1487 AACUUAUUGG 487 AAcuuAuuGGA 488 UuAcAACCUCc AD- AGGUUGUAA GGuuGuAATsT AAuAAGUUTsT 12291 3228-3246 1488 CUGGAGAGGU 489 cuGGAGAGGuc 490 CcACUUuAGAC AD- CUAAAGUGG uAAAGuGGTsT CUCUCcAGTsT 12292 3703-3721 1489 AAAAAAGAUA 491 AAAAAAGAuAu 492 ACUGCCUuAuA AD- UAAGGCAGU AAGGcAGuTsT UCUUUUUUTsT 12293 3737-3755 1490 GAAUUUUGAU 493 GAAuuuuGAuA 494 UGGGuAGAuAU AD- AUCUACCCA ucuAcccATsT cAAAAUUCTsT 12294 4573-4591 1491 GUAUUUUUGA 495 GuAuuuuuGAu 496 GUUGCcAGAUc AD- UCUGGCAAC cuGGcAAcTsT AAAAAuACTsT 12295 526-544 1492 AGGAUCCCUU 497 AGGAucccuuG 498 AuACcAGCcAA AD- GGCUGGUAU GcuGGuAuTsT GGGAUCCUTsT 12296 527-545 1493 GGAUCCCUUG 499 GGAucccuuGG 500 uAuACcAGCcA AD- GCUGGUAUA cuGGuAuATsT AGGGAUCCTsT 12297 256-274 1494 CAAUAGUAGA 501 cAAuAGuAGAA 502 GGAUcAcAUUC AD- AUGUGAUCC uGuGAuccTsT uACuAUUGTsT 12298 427-445 1495 GCUAUAAUUG 503 GcuAuAAuuGc 504 AAGAuAGUGcA AD- CACUAUCUU AcuAucuuTsT AUuAuAGCTsT 12299 554-572 1496 UACCCUUCAU 505 uAcccuucAuc 506 AAAAAUUUGAU AD- CAAAUUUUU AAAuuuuuTsT GAAGGGuATsT 12300 1210-1228 1497 AGAACAUAUU 507 AGAAcAuAuuG 508 GGCUuAUUcAA AD- GAAUAAGCC AAuAAGccTsT uAUGUUCUTsT 12301 1414-1432 1498 AAAUUGGUGC 509 AAAuuGGuGcu 510 UCCUcAAcAGc AD- UGUUGAGGA GuuGAGGATsT ACcAAUUUTsT 12302 1438-1456 1499 UGAAUAGGGU 511 uGAAuAGGGuu 512 AACUCUGuAAC AD- UACAGAGUU AcAGAGuuTsT CCuAUUcATsT 12303 1516-1534 1500 AAGAACUUGA 513 AAGAAcuuGAA 514 UGAGUGGUUUc AD- AACCACUCA AccAcucATsT AAGUUCUUTsT 12304 2279-2297 1501 AAUAAAGCAG 515 AAuAAAGcAGA 516 GGAAUGGGUCU AD- ACCCAUUCC cccAuuccTsT GCUUuAUUTsT 12305 2939-2957 1502 AUACCCAUCA 517 AuAcccAucAA 518 uACcAGUGUUG AD- ACACUGGUA cAcuGGuATsT AUGGGuAUTsT 12306 3142-3160 1503 UGGAUUGUUC 519 uGGAuuGuucA 520 CcAAUUGAUGA AD- AUCAAUUGG ucAAuuGGTsT AcAAUCcATsT 12307 3229-3247 1504 UGGAGAGGUC 521 uGGAGAGGucu 522 UCcACUUuAGA AD- UAAAGUGGA AAAGuGGATsT CCUCUCcATsT 12308 3763-3781 1505 GUCAUCCCUA 523 GucAucccuAu 524 AGUGAACuAuA AD- UAGUUCACU AGuucAcuTsT GGGAUGACTsT 12309 4801-4819 1506 AUAAUGGCUA 525 AuAAuGGcuAu 526 GAGAAAUuAuA AD- UAAUUUCUC AAuuucucTsT GCcAUuAUTsT 12310 529-547 1507 AUCCCUUGGC 527 AucccuuGGcu 528 AUuAuACcAGC AD- UGGUAUAAU GGuAuAAuTsT cAAGGGAUTsT 12311 425-443 1508 GGGCUAUAAU 529 GGGcuAuAAuu 530 GAuAGUGcAAU AD- UGCACUAUC GcAcuAucTsT uAuAGCCCTsT 12312 1104-1122 1509 GAUUCUCUUG 531 GAuucucuuGG 532 uACGCCCUCcA AD- GAGGGCGUA AGGGcGuATsT AGAGAAUCTsT 12313 1155-1173 1510 GCAUCUCUCA 533 GcAucucucAA 534 CCUcAAGAUUG AD- AUCUUGAGG ucuuGAGGTsT AGAGAUGCTsT 12314 2403-2421 1511 CAGCAGAAAU 535 cAGcAGAAAuc 536 uAUCCUuAGAU AD- CUAAGGAUA uAAGGAuATsT UUCUGCUGTsT 12315 3115-3133 1512 GUCAAGAGCC 537 GucAAGAGccA 538 UCuAcAGAUGG AD- AUCUGUAGA ucuGuAGATsT CUCUUGACTsT 12316 3209-3227 1513 AAACAGAGGC 539 AAAcAGAGGcA 540 UGUGUuAAUGC AD- AUUAACACA uuAAcAcATsT CUCUGUUUTsT 12317 3293-3311 1514 AGCCCAGAUC 541 AGcccAGAucA 542 UuAAAGGUUGA AD- AACCUUUAA AccuuuAATsT UCUGGGCUTsT 12318 4574-4592 1515 UAUUUUUGAU 543 uAuuuuuGAuc 544 GGUUGCcAGAU AD- CUGGCAACC uGGcAAccTsT cAAAAAuATsT 12319 352-370 1516 UGUUUGGAGC 545 uGuuuGGAGcA 546 UuAGuAGAUGC AD- AUCUACUAA ucuAcuAATsT UCcAAAcATsT 12320 741-759 1517 GAAAUUACAG 547 GAAAuuAcAGu 548 UGUUGUGuACU AD- UACACAACA AcAcAAcATsT GuAAUUUCTsT 12321 1478-1496 1518 ACUUGACCAG 549 AcuuGAccAGu 550 AGAUUuAcACU AD- UGUAAAUCU GuAAAucuTsT GGUcAAGUTsT 12322 1483-1501 1519 ACCAGUGUAA 551 AccAGuGuAAA 552 AGGUcAGAUUu AD- AUCUGACCU ucuGAccuTsT AcACUGGUTsT 12323 1967-1985 1520 AGAACAAUCA 553 AGAAcAAucAu 554 UGCUGCuAAUG AD- UUAGCAGCA uAGcAGcATsT AUUGUUCUTsT 12324 2247-2265 1521 CAAUGUGGAA 555 cAAuGuGGAAA 556 cAGUuAGGUUU AD- ACCUAACUG ccuAAcuGTsT CcAcAUUGTsT 12325 2500-2518 1522 ACCAAGAAGG 557 AccAAGAAGGu 558 AAUUUUGuACC AD- UACAAAAUU AcAAAAuuTsT UUCUUGGUTsT 12326 2508-2526 1523 GGUACAAAAU 559 GGuAcAAAAuu 560 CUUcAACcAAU AD- UGGUUGAAG GGuuGAAGTsT UUUGuACCTsT 12327 3138-3156 1524 GGUGUGGAUU 561 GGuGuGGAuuG 562 UUGAUGAAcAA AD- GUUCAUCAA uucAucAATsT UCcAcACCTsT 12328 4304-4322 1525 AGAGUUCACA 563 AGAGuucAcAA 564 UGGGCUUUUUG AD- AAAAGCCCA AAAGcccATsT UGAACUCUTsT 12329 4711-4729 1526 UGAUAGCUAA 565 uGAuAGcuAAA 566 UGGUUuAAUUu AD- AUUAAACCA uuAAAccATsT AGCuAUcATsT 12330 1221-1239 1527 AAUAAGCCUG 567 AAuAAGccuGA 568 GAUUcACUUcA AD- AAGUGAAUC AGuGAAucTsT GGCUuAUUTsT 12331 1705-1723 1528 CAGUUGACCA 569 cAGuuGAccAA 570 GcAUUGUGUUG AD- ACACAAUGC cAcAAuGcTsT GUcAACUGTsT 12332 3137-3155 1529 UGGUGUGGAU 571 uGGuGuGGAuu 572 UGAUGAAcAAU AD- UGUUCAUCA GuucAucATsT CcAcACcATsT 12333 4292-4310 1530 AUUCACCCUG 573 AuucAcccuGA 574 GAACUCUGUcA AD- ACAGAGUUC cAGAGuucTsT GGGUGAAUTsT 12334 1829-1847 1531 UAAGACCUUA 575 uAAGAccuuAu 576 AUUACcAAAuA AD- UUUGGUAAU uuGGuAAuTsT AGGUCUuATsT 12335 2244-2262 1532 AAGCAAUGUG 577 AAGcAAuGuGG 578 UuAGGUUUCcA AD- GAAACCUAA AAAccuAATsT cAUUGCUUTsT 12336 2888-2906 1533 UCUGAAACUG 579 ucuGAAAcuGG 580 UGGGAuAUCcA AD- GAUAUCCCA AuAucccATsT GUUUcAGATsT 12337 TABLE 2B 1st single SDs 1st 2nd single SDs 2nd SDs 3^(rd) dose screen screen dose screen screen screen @ 50 nM (among @ 25 nM (among 3rd single (among duplex [% resudual quadru- [% resudual quadru- dose screen quadru- name mRNA] plicates) mRNA] plicates) @ 25 nM plicates) AD-12072  65%  2%  82%   5% AD-12073  84%  1%  61%   6% AD-12074  51%  3%  36%   9% AD-12075  56%  4%  36%   4% AD-12076  21%  4%  13%   3% AD-12077  11%  2%   6%   1% AD-12078  22%  3%   9%   2% AD-12079  22% 10%  15%   7% AD-12080  68%  4%  52%  13% AD-12081  34%  8%  35%  24% AD-12082  20%  2%  92%   5% AD-12083  85%  6%  63%  10% AD-12084  18%  6%  17%   4% AD-12085  13%  4%  12%   4% AD-12086  26%  5%  17%   3% AD-12087  95%  4%  80%   4% AD-12088  29%  6%  29%   2% AD-12089  69%  5%  64%   7% AD-12090  46% 15%  34%   5% AD-12091  16%  6%  17%   3% AD-12092  82% 26%  63%   5% AD-12093  84%  4%  70%   4% AD-12094  46%  3%  34%   1% AD-12095  14%  2%  13%   1% AD-12096  26% 11%  17%   1% AD-12097  23%  2%  21%   1% AD-12098  41% 14%  17%   3% AD-12099  57%  2%  48%   6% AD-12100 101% 11%  98%   8% AD-12101  46%  7%  32%   2% AD-12102  96% 17%  88%  18% AD-12103  19%  5%  20%   2% AD-12104  40%  8%  24%   2% AD-12105  39%  2%  36%  10% AD-12106  87%  6%  79%  19% AD-12107  29%  2%  32%  16% AD-12108  38%  4%  39%   8% AD-12109  49%  3%  44%  10% AD-12110  85%  5%  80%  14% AD-12111  64%  6%  71%  18% AD-12112  48%  4%  41%   5% AD-12113  13%  0%  14%   3% AD-12114  32%  6%  16%   4% AD-12115   8%  4%   7%   5% AD-12116  74%  5%  61%   7% AD-12117  21%  4%  20%   2% AD-12118  44%  4%  42%   6% AD-12119  37%  4%  24%   3% AD-12120  22%  2%  15%   4% AD-12121  32%  1%  22%   2% AD-12122  36% 16%  19%   5% AD-12123  28%  1%  16% AD-12124  28%  2%  16% AD-12125  15%  1%  14% AD-12126  51% 22%  27% AD-12127  54%  4%  42%   9% AD-12128  29%  1%  20%   2% AD-12129  22%  3%  19%   3% AD-12130  53%  6%  42%   7% AD-12131  28%  5%  22%   3% AD-12132  88%  2%  90%  18% AD-12133  34%  2%  26%   6% AD-12134  18%  3%  14%   2% AD-12135  50%  6%  37%   4% AD-12136  42% 19%  22%   2% AD-12137  85% 12%  92%   4% AD-12138  47%  6%  49%   1% AD-12139  80%  5%  72%   4% AD-12140  97% 22%  67%   9% AD-12141 120%  4% 107%  10% AD-12142  55%  8%  33%   4% AD-12143  64% 34%  19%   2% AD-12144  58% 29%  17%   2% AD-12145  27%  8%  18%   2% AD-12146  19% 20%  15%   1% AD-12147  29%  9%  35%   3% AD-12148  30%  3%  56%   5% AD-12149   8%  2%  12%   3% AD-12150  31%  2%  31%   7% AD-12151   9%  5%  14%   2% AD-12152   3%  3%  23%   3% AD-12153  20%  6%  34%   4% AD-12154  24%  7%  44%   3% AD-12155  33%  6%  53%  11% AD-12156  35%  5%  40%   5% AD-12157   8%  3%  23%   4% AD-12158  13%  2%  22%   5% AD-12159  34%  6%  46%   5% AD-12160  19%  3%  31%   4% AD-12161  88%  4%  83%   7% AD-12162  26%  7%  32%   7% AD-12163  55%  9%  40%   3% AD-12164  21%   3% AD-12165  30%  3%  41%   4% AD-12166   9% 10%  22%   9% AD-12167  26%  3%  30%   2% AD-12168  54%  4%  59%  20% AD-12169  41%  4%  51%  16% AD-12170  43%  4%  52%  20% AD-12171  67%  3%  73%  25% AD-12172  53% 15%  37%   2% AD-12173  39%  0%  39%   0% AD-12174  41%  5%  27%   0% AD-12175  29%  0%  38%  14% AD-12176  43%  2%  56%  25% AD-12177  68%  6%  74%  30% AD-12178  41%  4%  41%   6% AD-12179  53%  5%  44%   5% AD-12180  16%  2%  13%   4% AD-12181  19%  3%  14%   2% AD-12182  16%  4%  18%   8% AD-12183  26%  3%  19%   4% AD-12184  54%  2%  77%   8% AD-12185   8%  1%   9%   1% AD-12186  36%  3%  41%   6% AD-12187  34% 17%  27%   1% AD-12188  30%  3%  27%   4% AD-12189  51%  4%  48%   5% AD-12190  33%  2%  26%   4% AD-12191  20%  2%  13%   0% AD-12192  21%  1%  23%  10% AD-12193  64%  8%  98%   6% AD-12194   8%  2%  15%   4% AD-12195  34%  2%  48%   3% AD-12196  34%  2%  51%   3% AD-12197  75%  4%  93%   6% AD-12198  55%  5%  48%   2% AD-12199 102%  6% 118%   9% AD-12200  75%  6%  60%  12% AD-12201  42%  3%  16%   4% AD-12202  29%  4%   9%   3% AD-12203 114% 14%  89%  20% AD-12204  64%  7%  26%   5% AD-12205  66% 12%  35%   4% AD-12206  46%  3%  32%  12% AD-12207  57%  5%  40%   6% AD-12208  30%  8%  10%   5% AD-12209 101%  6% 102%  23% AD-12210  38% 11%  27%  14% AD-12211  16%  6%  10%   5% AD-12212  59%  8%  65%   5% AD-12213  24%  9%  12%   2% AD-12214  67% 14%  70%  12% AD-12215  29% 13%  13%   4% AD-12216  36%  4%  13%   1% AD-12217  36%  9%  11%   2% AD-12218  35%  5%  17%   3% AD-12219  41%  9%  14%   1% AD-12220  37%  5%  23%   3% AD-12221  58%  7%  39%   6% AD-12222  74%  9%  53%   3% AD-12223  74% 10%  67%   7% AD-12224  24%  2%  11%   2% AD-12225  75%  5%  76%  14% AD-12226  45%  8%  40%   3% AD-12227  61%  6%  47%   5% AD-12228  28%  3%  25%   5% AD-12229  54% 13%  37%   6% AD-12230  70% 17%  65%   4% AD-12231  32% 12%  22%   6% AD-12232  30%  3%  17%   2% AD-12233  38%  2%  32%   3% AD-12234  90%  5%  95%   7% AD-12235  57%  7%  46%   3% AD-12236  34%  8%  16%   2% AD-12237  42%  9%  32%   8% AD-12238  42%  6%  34%   6% AD-12239  42%  3%  40%   4% AD-12240  47%  6%  36%   5% AD-12241  69%  5%  70%   8% AD-12242  61%  2%  47%   3% AD-12243  26%  7%  15%   1% AD-12244  25%  6%  15%   1% AD-12245  65%  6%  83%  13% AD-12246  29%  7%  31%   6% AD-12247  57% 13%  50%   3% AD-12248  36%  8%  20%   3%  15%  7% AD-12249  44%  3%  70%  11% 103% 34% AD-12250  47%  5%  18%   5%  17%  4% AD-12251 121% 28%  35%   8%  60% 42% AD-12252  94% 19%   8%   3%   5%  3% AD-12253  94% 33%  42%   8%  49% 27% AD-12254 101% 58%  70%   5%  80% 32% AD-12255 163% 27%  28%   6%  36% 10% AD-12256 112% 62%  18%   3%   9%  4% AD-12257  10%  4%   9%   2%   6%  2% AD-12258  27%  9%  18%   3%  20%  6% AD-12259  20%  5%  12%   2%  13%  5% AD-12260  22%  7%  81%   7%  65% 13% AD-12261 122% 11%  66%   7%  80% 22% AD-12262  97% 30%  33%   6%  44% 18% AD-12263 177% 57%  85%  11%  84% 15% AD-12264  37%  6%  10%   1%  10%  4% AD-12265  40%  8%  17%   1%  20% 10% AD-12266  33%  9%   9%   1%   8%  4% AD-12267  34% 13%  11%   1%   6%  2% AD-12268  34%  6%  11%   1%   9%  2% AD-12269  54%  6%  33%   4%  29%  7% AD-12270  52%  5%  29%   4%  27%  6% AD-12271  53%  7%  27%   3%  19%  6% AD-12272  85% 15%  57%   7%  51% 16% AD-12273  36%  6%  26%   2%  30%  5% AD-12274  75% 21%  40%   2%  50% 19% AD-12275  29%  9%   8%   1%   8%  4% AD-12276  45% 19%  15%   2%  16% 12% AD-12277  58% 17%  32%   2%  55% 14% AD-12278 120% 35%  96%  10% 124% 38% AD-12279  47% 29%  17%   1%  12%  4% AD-12280   2%  0%   3%   1% AD-12281   2%  0%   5%   2% AD-12282   3%  0%  25%   5% AD-12283   3%  1%  35%   4% AD-12284   5%  2%  49%   8% AD-12285   7%  7%  21%  26% AD-12286  28% 34%  12%   7% AD-12287  40% 21%  51%  23% AD-12288  26%  7% 155% 146% AD-12289  43% 21% 220% 131% AD-12290   2%  1%  81%  23% AD-12291   4%  1%  70%   3% AD-12292   2%  1%   6%   2% AD-12293   4%  2%  36%   3% AD-12294  10%  6%  38%   3% AD-12295  29% 31%  37%   3% AD-12296  82%  4%  89%   2% AD-12297  75%  3%  65%   2% AD-12298  73%  4%  60%   3% AD-12299  76%  4%  66%   4% AD-12300  36%  4%  15%   1% AD-12301  33%  4%  18%   2% AD-12302  66%  5%  65%   3% AD-12303  35%  6%  17%   2% AD-12304  70%  8%  70%   6% AD-12305  63%  8%  80%   7% AD-12306  23%  6%  20%   3% AD-12307  78% 10%  58%   5% AD-12308  27%  8%  15%   2% AD-12309  58% 11%  42%   3% AD-12310 106% 23%  80%   2% AD-12311  73% 12%  60%   2% AD-12312  39%  3%  36%   3% AD-12313  64%  9%  49%   6% AD-12314  28%  7%  14%   6% AD-12315  31%  7%  13%   2% AD-12316  42%  5%  14%   2% AD-12317  34%  9%  15%   5% AD-12318  46%  4%  28%   4% AD-12319  77%  3%  56%   4% AD-12320  55%  7%  41%   3% AD-12321  21%  3%  10%   2% AD-12322  27%  8%  30%  12% AD-12323  26%  7%  35%  18% AD-12324  27%  8%  27%  14% AD-12325  32% 12%  32%  22% AD-12326  42% 22%  45%  41% AD-12327  36% 14%  37%  32% AD-12328  45%  2%  31%   3% AD-12329  61%  4%  34%   3% AD-12330  63%  5%  38%   4% AD-12331  50%  2%  26%   5% AD-12332  80%  4%  51%   7% AD-12333  34%  6%  12%   2% AD-12334  27%  2%  18%   3% AD-12335  84%  6%  60%   7% AD-12336  45%  4%  36%   4% AD-12337  30%  7%  19%   2%

TABLE 3 single SDs dose 2nd screen @ screen SEQ SEQ 25 nM [% (among ID ID duplex residual  quadru- sequence (5′-3′) NO. sequence (5′-3′) NO. name mRNA] plicates) ccAuuAcuAcAGuAGcAcuTsT  582 AGUGCuACUGuAGuAAUGGTsT  583 AD-14085  19%  1% AucuGGcAAccAuAuuucuTsT  584 AGAAAuAUGGUUGCcAGAUTsT  585 AD-14086  38%  1% GAuAGcuAAAuuAAAccAATsT  586 UUGGUUuAAUUuAGCuAUCTsT  587 AD-14087  75% 10% AGAuAccAuuAcuAcAGuATsT  588 uACUGuAGuAAUGGuAUCUTsT  589 AD-14088  22%  8% GAuuGuucAucAAuuGGcGTsT  590 CGCcAAUUGAUGAAcAAUCTsT  591 AD-14089  70% 12% GcuuucuccucGGcucAcuTsT  592 AGuGAGCCGAGGAGAAAGCTsT  593 AD-14090  79% 11% GGAGGAuuGGcuGAcAAGATsT  594 UCUUGUcAGCcAAUCCUCCTsT  595 AD-14091  29%  3% uAAuGAAGAGuAuAccuGGTsT  596 CcAGGuAuACUCUUcAUuATsT  597 AD-14092  23%  2% uuucAccAAAccAuuuGuATsT  598 uAcAAAUGGUUUGGUGAAATsT  599 AD-14093  60%  2% cuuAuuAAGGAGuAuAcGGTsT  600 CCGuAuACUCCUuAAuAAGTsT  601 AD-14094  11%  3% GAAAucAGAuGGAcGuAAGTsT  602 CUuACGUCcAUCUGAUUUCTsT  603 AD-14095  10%  2% cAGAuGucAGcAuAAGcGATsT  604 UCGCUuAUGCUGAcAUCUGTsT  605 AD-14096  27%  2% AucuAAcccuAGuuGuAucTsT  606 GAuAcAACuAGGGUuAGAUTsT  607 AD-14097  45%  6% AAGAGcuuGuuAAAAucGGTsT  608 CCGAUUUuAAcAAGCUCUUTsT  609 AD-14098  50% 10% uuAAGGAGuAuAcGGAGGATsT  610 UCCUCCGuAuACUCCUuAATsT  611 AD-14099  12%  4% uuGcAAuGuAAAuAcGuAuTsT  612 AuACGuAUUuAcAUUGcAATsT  613 AD-14100  49%  7% ucuAAcccuAGuuGuAuccTsT  614 GGAuAcAACuAGGGUuAGATsT  615 AD-14101  36%  1% cAuGuAucuuuuucucGAuTsT  616 AUCGAGAAAAAGAuAcAUGTsT  617 AD-14102  49%  3% GAuGucAGcAuAAGcGAuGTsT  618 cAUCGCUuAUGCUGAcAUCTsT  619 AD-14103  74%  5% ucccAAcAGGuAcGAcAccTsT  620 GGUGUCGuACCUGUUGGGATsT  621 AD-14104  27%  3% uGcucAcGAuGAGuuuAGuTsT  622 ACuAAACUcAUCGUGAGcATsT  623 AD-14105  34%  4% AGAGcuuGuuAAAAucGGATsT  624 UCCGAUUUuAAcAAGCUCUTsT  625 AD-14106   9%  2% GcGuAcAAGAAcAucuAuATsT  626 uAuAGAUGUUCUUGuACGCTsT  627 AD-14107   5%  1% GAGGuuGuAAGccAAuGuuTsT  628 AAcAUUGGCUuAcAACCUCTsT  629 AD-14108  15%  1% AAcAGGuAcGAcAccAcAGTsT  630 CUGUGGUGUCGuACCUGUUTsT  631 AD-14109  91%  2% AAcccuAGuuGuAucccucTsT  632 GAGGGAuAcAACuAGGGUUTsT  633 AD-14110  66%  5% GcAuAAGcGAuGGAuAAuATsT  634 uAUuAUCcAUCGCUuAUGCTsT  635 AD-14111  33%  3% AAGcGAuGGAuAAuAccuATsT  636 uAGGuAUuAUCcAUCGCUUTsT  637 AD-14112  51%  3% uGAuccuGuAcGAAAAGAATsT  638 UUCUUUUCGuAcAGGAUcATsT  639 AD-14113  22%  3% AAAAcAuuGGccGuucuGGTsT  640 CcAGAACGGCcAAUGUUUUTsT  641 AD-14114 117%  8% cuuGGAGGGcGuAcAAGAATsT  642 UUCUUGuACGCCCUCcAAGTsT  643 AD-14115  50%  8% GGcGuAcAAGAAcAucuAuTsT  644 AuAGAUGUUCUUGuACGCCTsT  645 AD-14116  14%  3% AcucuGAGuAcAuuGGAAuTsT  646 AUUCcAAUGuACUcAGAGUTsT  647 AD-14117  12%  4% uuAuuAAGGAGuAuAcGGATsT  648 UCCGuAuACUCCUuAAuAATsT  649 AD-14118  26%  4% uAAGGAGuAuAcGGAGGAGTsT  650 CUCCUCCGuAuACUCCUuATsT  651 AD-14119  24%  5% AAAucAAuAGucAAcuAAATsT  652 UUuAGUUGACuAUUGAUUUTsT  653 AD-14120   8%  1% AAucAAuAGucAAcuAAAGTsT  654 CUUuAGUUGACuAUUGAUUTsT  655 AD-14121  24%  2% uucucAGuAuAcuGuGuAATsT  656 UuAcAcAGuAuACUGAGAATsT  657 AD-14122  10%  1% uGuGAAAcAcucuGAuAAATsT  658 UUuAUcAGAGUGUUUcAcATsT  659 AD-14123   8%  1% AGAuGuGAAucucuGAAcATsT  660 UGUUcAGAGAUUcAcAUCUTsT  661 AD-14124   9%  2% AGGuuGuAAGccAAuGuuGTsT  662 cAAcAUUGGCUuAcAACCUTsT  663 AD-14125 114%  6% uGAGAAAucAGAuGGAcGuTsT  664 ACGUCcAUCUGAUUUCUcATsT  665 AD-14126   9%  1% AGAAAucAGAuGGAcGuAATsT  666 UuACGUCcAUCUGAUUUCUTsT  667 AD-14127  57%  6% AuAucccAAcAGGuAcGAcTsT  668 GUCGuACCUGUUGGGAuAUTsT  669 AD-14128 104%  6% cccAAcAGGuAcGAcAccATsT  670 UGGUGUCGuACCUGUUGGGTsT  671 AD-14129  21%  2% AGuAuAcuGAAGAAccucuTsT  672 AGAGGUUCUUcAGuAuACUTsT  673 AD-14130  57%  6% AuAuAuAucAGccGGGcGcTsT  674 GCGCCCGGCUGAuAuAuAUTsT  675 AD-14131  93%  6% AAucuAAcccuAGuuGuAuTsT  676 AuAcAACuAGGGUuAGAUUTsT  677 AD-14132  75%  8% cuAAcccuAGuuGuAucccTsT  678 GGGAuAcAACuAGGGUuAGTsT  679 AD-14133  66%  4% cuAGuuGuAucccuccuuuTsT  680 AAAGGAGGGAuAcAACuAGTsT  681 AD-14134  44%  6% AGAcAucuGAcuAAuGGcuTsT  682 AGCcAUuAGUcAGAUGUCUTsT  683 AD-14135  55%  6% GAAGcucAcAAuGAuuuAATsT  684 UuAAAUcAUUGUGAGCUUCTsT  685 AD-14136  29%  3% AcAuGuAucuuuuucucGATsT  686 UCGAGAAAAAGAuAcAUGUTsT  687 AD-14137  40%  3% ucGAuucAAAucuuAAcccTsT  688 GGGUuAAGAUUUGAAUCGATsT  689 AD-14138  39%  5% ucuuAAcccuuAGGAcucuTsT  690 AGAGUCCuAAGGGUuAAGATsT  691 AD-14139  71% 11% GcucAcGAuGAGuuuAGuGTsT  692 cACuAAACUcAUCGUGAGCTsT  693 AD-14140  43% 15% cAuAAGcGAuGGAuAAuAcTsT  694 GuAUuAUCcAUCGCUuAUGTsT  695 AD-14141  33%  6% AuAAGcGAuGGAuAAuAccTsT  696 GGuAUuAUCcAUCGCUuAUTsT  697 AD-14142  51% 14% ccuAAuAAAcuGcccucAGTsT  698 CUGAGGGcAGUUuAUuAGGTsT  699 AD-14143  42%  1% ucGGAAAGuuGAAcuuGGuTsT  700 ACcAAGUUcAACUUUCCGATsT  701 AD-14144   4%  4% GAAAAcAuuGGccGuucuGTsT  702 cAGAACGGCcAAUGUUUUCTsT  703 AD-14145  92%  5% AAGAcuGAucuucuAAGuuTsT  704 AACUuAGAAGAUcAGUCUUTsT  705 AD-14146  13%  2% GAGcuuGuuAAAAucGGAATsT  706 UUCCGAUUUuAAcAAGCUCTsT  707 AD-14147   8%  1% AcAuuGGccGuucuGGAGcTsT  708 GCUCcAGAACGGCcAAUGUTsT  709 AD-14148  80%  7% AAGAAcAucuAuAAuuGcATsT  710 UGcAAUuAuAGAUGUUCUUTsT  711 AD-14149  44%  7% AAAuGuGucuAcucAuGuuTsT  712 AAcAUGAGuAGAcAcAUUUTsT  713 AD-14150  32% 29% uGucuAcucAuGuuucucATsT  714 UGAGAAAcAUGAGuAGAcATsT  715 AD-14151  75% 11% GuAuAcuGuGuAAcAAucuTsT  716 AGAUUGUuAcAcAGuAuACTsT  717 AD-14152   8%  5% uAuAcuGuGuAAcAAucuATsT  718 uAGAUUGUuAcAcAGuAuATsT  719 AD-14153  17% 11% cuuAGuAGuGuccAGGAAATsT  720 UUUCCUGGAcACuACuAAGTsT  721 AD-14154  16%  4% ucAGAuGGAcGuAAGGcAGTsT  722 CUGCCUuACGUCcAUCUGATsT  723 AD-14155  11%  1% AGAuAAAuuGAuAGcAcAATsT  724 UUGUGCuAUcAAUUuAUCUTsT  725 AD-14156  10%  1% cAAcAGGuAcGAcAccAcATsT  726 UGUGGUGUCGuACCUGUUGTsT  727 AD-14157  29%  3% uGcAAuGuAAAuAcGuAuuTsT  728 AAuACGuAUUuAcAUUGcATsT  729 AD-14158  51%  3% AGucAGAAuuuuAucuAGATsT  730 UCuAGAuAAAAUUCUGACUTsT  731 AD-14159  53%  5% cuAGAAAucuuuuAAcAccTsT  732 GGUGUuAAAAGAUUUCuAGTsT  733 AD-14160  40%  3% AAuAAAucuAAcccuAGuuTsT  734 AACuAGGGUuAGAUUuAUUTsT  735 AD-14161  83%  7% AAuuuucuGcucAcGAuGATsT  736 UcAUCGUGAGcAGAAAAUUTsT  737 AD-14162  44%  6% GcccucAGuAAAuccAuGGTsT  738 CcAUGGAUUuACUGAGGGCTsT  739 AD-14163  57%  3% AcGuuuAAAAcGAGAucuuTsT  740 AAGAUCUCGUUUuAAACGUTsT  741 AD-14164   4%  1% AGGAGAuAGAAcGuuuAAATsT  742 UUuAAACGUUCuAUCUCCUTsT  743 AD-14165  11%  1% GAccGucAuGGcGucGcAGTsT  744 CUGCGACGCcAUGACGGUCTsT  745 AD-14166  90%  5% AccGucAuGGcGucGcAGcTsT  746 GCUGCGACGCcAUGACGGUTsT  747 AD-14167  49%  1% GAAcGuuuAAAAcGAGAucTsT  748 GAUCUCGUUUuAAACGUUCTsT  749 AD-14168  12%  2% uuGAGcuuAAcAuAGGuAATsT  750 UuACCuAUGUuAAGCUcAATsT  751 AD-14169  66%  4% AcuAAAuuGAucucGuAGATsT  752 UCuACGAGAUcAAUUuAGUTsT  753 AD-14170  52%  6% ucGuAGAAuuAucuuAAuATsT  754 uAUuAAGAuAAUUCuACGATsT  755 AD-14171  42%  4% GGAGAuAGAAcGuuuAAAATsT  756 UUUuAAACGUUCuAUCUCCTsT  757 AD-14172   3%  1% AcAAcuuAuuGGAGGuuGuTsT  758 AcAACCUCcAAuAAGUUGUTsT  759 AD-14173  29%  2% uGAGcuuAAcAuAGGuAAATsT  760 UUuACCuAUGUuAAGCUcATsT  761 AD-14174  69%  2% AucucGuAGAAuuAucuuATsT  762 uAAGAuAAUUCuACGAGAUTsT  763 AD-14175  53%  3% cuGcGuGcAGucGGuccucTsT  764 GAGGACCGACUGcACGcAGTsT  765 AD-14176 111%  4% cAcGcAGcGcccGAGAGuATsT  766 uACUCUCGGGCGCUGCGUGTsT  767 AD-14177  87%  6% AGuAccAGGGAGAcuccGGTsT  768 CCGGAGUCUCCCUGGuACUTsT  769 AD-14178  59%  2% AcGGAGGAGAuAGAAcGuuTsT  770 AACGUUCuAUCUCCUCCGUTsT  771 AD-14179   9%  2% AGAAcGuuuAAAAcGAGAuTsT  772 AUCUCGUUUuAAACGUUCUTsT  773 AD-14180  43%  2% AAcGuuuAAAAcGAGAucuTsT  774 AGAUCUCGUUUuAAACGUUTsT  775 AD-14181  70% 10% AGcuuGAGcuuAAcAuAGGTsT  776 CCuAUGUuAAGCUcAAGCUTsT  777 AD-14182 100%  7% AGcuuAAcAuAGGuAAAuATsT  778 uAUUuACCuAUGUuAAGCUTsT  779 AD-14183  60%  5% uAGAGcuAcAAAAccuAucTsT  780 GAuAGGUUUUGuAGCUCuATsT  781 AD-14184 129%  6% uAGuuGuAucccuccuuuATsT  782 uAAAGGAGGGAuAcAACuATsT  783 AD-14185  62%  4% AccAcccAGAcAucuGAcuTsT  784 AGUcAGAUGUCUGGGUGGUTsT  785 AD-14186  42%  3% AGAAAcuAAAuuGAucucGTsT  786 CGAGAUcAAUUuAGUUUCUTsT  787 AD-14187 123% 12% ucucGuAGAAuuAucuuAATsT  788 UuAAGAuAAUUCuACGAGATsT  789 AD-14188  38%  2% cAAcuuAuuGGAGGuuGuATsT  790 uAcAACCUCcAAuAAGUUGTsT  791 AD-14189  13%  1% uuGuAucccuccuuuAAGuTsT  792 ACUuAAAGGAGGGAuAcAATsT  793 AD-14190  59%  3% ucAcAAcuuAuuGGAGGuuTsT  794 AACCUCcAAuAAGUUGUGATsT  795 AD-14191  93%  3% AGAAcuGuAcucuucucAGTsT  796 CUGAGAAGAGuAcAGUUCUTsT  797 AD-14192  45%  5% GAGcuuAAcAuAGGuAAAuTsT  798 AUUuACCuAUGUuAAGCUCTsT  799 AD-14193  57%  3% cAccAAcAucuGuccuuAGTsT  800 CuAAGGAcAGAUGUUGGUGTsT  801 AD-14194  51%  4% AAAGcccAcuuuAGAGuAuTsT  802 AuACUCuAAAGUGGGCUUUTsT  803 AD-14195  77%  5% AAGcccAcuuuAGAGuAuATsT  804 uAuACUCuAAAGUGGGCUUTsT  805 AD-14196  42%  6% GAccuuAuuuGGuAAucuGTsT  806 cAGAUuACcAAAuAAGGUCTsT  807 AD-14197  15%  2% GAuuAAuGuAcucAAGAcuTsT  808 AGUCUUGAGuAcAUuAAUCTsT  809 AD-14198  12%  2% cuuuAAGAGGccuAAcucATsT  810 UGAGUuAGGCCUCUuAAAGTsT  811 AD-14199  18%  2% uuAAAccAAAcccuAuuGATsT  812 UcAAuAGGGUUUGGUUuAATsT  813 AD-14200  72%  9% ucuGuuGGAGAucuAuAAuTsT  814 AUuAuAGAUCUCcAAcAGATsT  815 AD-14201   9%  3% cuGAuGuuucuGAGAGAcuTsT  816 AGUCUCUcAGAAAcAUcAGTsT  817 AD-14202  25%  3% GcAuAcucuAGucGuucccTsT  818 GGGAACGACuAGAGuAUGCTsT  819 AD-14203  21%  1% GuuccuuAucGAGAAucuATsT  820 uAGAUUCUCGAuAAGGAACTsT  821 AD-14204   4%  2% GcAcuuGGAucucucAcAuTsT  822 AUGUGAGAGAUCcAAGUGCTsT  823 AD-14205   5%  1% AAAAAAGGAAcuAGAuGGcTsT  824 GCcAUCuAGUUCCUUUUUUTsT  825 AD-14206  79%  6% AGAGcAGAuuAccucuGcGTsT  826 CGcAGAGGuAAUCUGCUCUTsT  827 AD-14207  55%  2% AGcAGAuuAccucuGcGAGTsT  828 CUCGcAGAGGuAAUCUGCUTsT  829 AD-14208 100%  4% cccuGAcAGAGuucAcAAATsT  830 UUUGUGAACUCUGUcAGGGTsT  831 AD-14209  34%  3% GuuuAccGAAGuGuuGuuuTsT  832 AAAcAAcACUUCGGuAAACTsT  833 AD-14210  13%  2% uuAcAGuAcAcAAcAAGGATsT  834 UCCUUGUUGUGuACUGuAATsT  835 AD-14211   9%  1% AcuGGAucGuAAGAAGGcATsT  836 UGCCUUCUuACGAUCcAGUTsT  837 AD-14212  20%  3% GAGcAGAuuAccucuGcGATsT  838 UCGcAGAGGuAAUCUGCUCTsT  839 AD-14213  48%  5% AAAAGAAGuuAGuGuAcGATsT  840 UCGuAcACuAACUUCUUUUTsT  841 AD-14214  28% 18% GAccAuuuAAuuuGGcAGATsT  842 UCUGCcAAAUuAAAUGGUCTsT  843 AD-14215 132%  0% GAGAGGAGuGAuAAuuAAATsT  844 UUuAAUuAUcACUCCUCUCTsT  845 AD-14216   3%  0% cuGGAGGAuuGGcuGAcAATsT  846 UUGUcAGCcAAUCCUCcAGTsT  847 AD-14217  19%  1% cucuAGucGuucccAcucATsT  848 UGAGUGGGAACGACuAGAGTsT  849 AD-14218  67%  8% GAuAccAuuAcuAcAGuAGTsT  850 CuACUGuAGuAAUGGuAUCTsT  851 AD-14219  76%  4% uucGucuGcGAAGAAGAAATsT  852 UUUCUUCUUCGcAGACGAATsT  853 AD-14220  33%  8% GAAAAGAAGuuAGuGuAcGTsT  854 CGuAcACuAACUUCUUUUCTsT  855 AD-14221  25%  2% uGAuGuuuAccGAAGuGuuTsT  856 AAcACUUCGGuAAAcAUcATsT  857 AD-14222   7%  2% uGuuuGuccAAuucuGGAuTsT  858 AUCcAGAAUUGGAcAAAcATsT  859 AD-14223  19%  2% AuGAAGAGuAuAccuGGGATsT  860 UCCcAGGuAuACUCUUcAUTsT  861 AD-14224  13%  1% GcuAcucuGAuGAAuGcAuTsT  862 AUGcAUUcAUcAGAGuAGCTsT  863 AD-14225  15%  2% GcccuuGuAGAAAGAAcAcTsT  864 GUGUUCUUUCuAcAAGGGCTsT  865 AD-14226  11%  0% ucAuGuuccuuAucGAGAATsT  866 UUCUCGAuAAGGAAcAUGATsT  867 AD-14227   5%  1% GAAuAGGGuuAcAGAGuuGTsT  868 cAACUCUGuAACCCuAUUCTsT  869 AD-14228  34%  3% cAAAcuGGAucGuAAGAAGTsT  870 CUUCUuACGAUCcAGUUUGTsT  871 AD-14229  15%  2% cuuAuuuGGuAAucuGcuGTsT  872 cAGcAGAUuACcAAAuAAGTsT  873 AD-14230  20%  1% AGcAAuGuGGAAAccuAAcTsT  874 GUuAGGUUUCcAcAUUGCUTsT  875 AD-14231  18%  1% AcAAuAAAGcAGAcccAuuTsT  876 AAUGGGUCUGCUUuAUUGUTsT  877 AD-14232  21%  1% AAccAcuuAGuAGuGuccATsT  878 UGGAcACuACuAAGUGGUUTsT  879 AD-14233 106% 12% AGucAAGAGccAucuGuAGTsT  880 CuAcAGAUGGCUCUUGACUTsT  881 AD-14234  35%  3% cucccuAGAcuucccuAuuTsT  882 AAuAGGGAAGUCuAGGGAGTsT  883 AD-14235  48%  4% AuAGcuAAAuuAAAccAAATsT  884 UUUGGUUuAAUUuAGCuAUTsT  885 AD-14236  23%  3% uGGcuGGuAuAAuuccAcGTsT  886 CGUGGAAUuAuACcAGCcATsT  887 AD-14237  79%  9% uuAuuuGGuAAucuGcuGuTsT  888 AcAGcAGAUuACcAAAuAATsT  889 AD-14238  92%  7% AAcuAGAuGGcuuucucAGTsT  890 CUGAGAAAGCcAUCuAGUUTsT  891 AD-14239  20%  2% ucAuGGcGucGcAGccAAATsT  892 UUUGGCUGCGACGCcAUGATsT  893 AD-14240  71%  6% AcuGGAGGAuuGGcuGAcATsT  894 UGUcAGCcAAUCCUCcAGUTsT  895 AD-14241  14%  1% cuAuAAuuGcAcuAucuuuTsT  896 AAAGAuAGUGcAAUuAuAGTsT  897 AD-14242  11%  2% AAAGGucAccuAAuGAAGATsT  898 UCUUcAUuAGGUGACCUUUTsT  899 AD-14243  11%  1% AuGAAuGcAuAcucuAGucTsT  900 GACuAGAGuAUGcAUUcAUTsT  901 AD-14244  15%  2% AAcAuAuuGAAuAAGccuGTsT  902 cAGGCUuAUUcAAuAUGUUTsT  903 AD-14245  80%  7% AAGAAGGcAGuuGAccAAcTsT  904 GUUGGUcAACUGCCUUCUUTsT  905 AD-14246  57%  5% GAuAcuAAAAGAAcAAucATsT  906 UGAUUGUUCUUUuAGuAUCTsT  907 AD-14247   9%  3% AuAcuGAAAAucAAuAGucTsT  908 GACuAUUGAUUUUcAGuAUTsT  909 AD-14248  39%  4% AAAAAGGAAcuAGAuGGcuTsT  910 AGCcAUCuAGUUCCUUUUUTsT  911 AD-14249  64%  2% GAAcuAGAuGGcuuucucATsT  912 UGAGAAAGCcAUCuAGUUCTsT  913 AD-14250  18%  2% GAAAccuAAcuGAAGAccuTsT  914 AGGUCUUcAGUuAGGUUUCTsT  915 AD-14251  56%  6% uAcccAucAAcAcuGGuAATsT  916 UuACcAGUGUUGAUGGGuATsT  917 AD-14252  48%  6% AuuuuGAuAucuAcccAuuTsT  918 AAUGGGuAGAuAUcAAAAUTsT  919 AD-14253  39%  5% AucccuAuAGuucAcuuuGTsT  920 cAAAGUGAACuAuAGGGAUTsT  921 AD-14254  44%  8% AuGGGcuAuAAuuGcAcuATsT  922 uAGUGcAAUuAuAGCCcAUTsT  923 AD-14255 108%  8% AGAuuAccucuGcGAGcccTsT  924 GGGCUCGcAGAGGuAAUCUTsT  925 AD-14256 108%  6% uAAuuccAcGuAcccuucATsT  926 UGAAGGGuACGUGGAAUuATsT  927 AD-14257  23%  2% GucGuucccAcucAGuuuuTsT  928 AAAACuGAGuGGGAACGACTsT  929 AD-14258  21%  3% AAAucAAucccuGuuGAcuTsT  930 AGUcAAcAGGGAUUGAUUUTsT  931 AD-14259  19%  2% ucAuAGAGcAAAGAAcAuATsT  932 uAUGUUCUUUGCUCuAUGATsT  933 AD-14260  10%  1% uuAcuAcAGuAGcAcuuGGTsT  934 CcAAGUGCuACUGuAGuAATsT  935 AD-14261  76%  3% AuGuGGAAAccuAAcuGAATsT  936 UUcAGUuAGGUUUCcAcAUTsT  937 AD-14262  13%  2% uGuGGAAAccuAAcuGAAGTsT  938 CUUcAGUuAGGUUUCcAcATsT  939 AD-14263  14%  2% ucuuccuuAAAuGAAAGGGTsT  940 CCCUUUcAUUuAAGGAAGATsT  941 AD-14264  65%  3% uGAAGAAccucuAAGucAATsT  942 UUGACUuAGAGGUUCUUcATsT  943 AD-14265  13%  1% AGAGGucuAAAGuGGAAGATsT  944 UCUUCcACUUuAGACCUCUTsT  945 AD-14266  18%  3% AuAucuAcccAuuuuucuGTsT  946 cAGAAAAAUGGGuAGAuAUTsT  947 AD-14267  50%  9% uAAGccuGAAGuGAAucAGTsT  948 CUGAUUcACUUcAGGCUuATsT  949 AD-14268  13%  3% AGAuGcAGAccAuuuAAuuTsT  950 AAUuAAAUGGUCUGcAUCUTsT  951 AD-14269  19%  4% AGuGuuGuuuGuccAAuucTsT  952 GAAUUGGAcAAAcAAcACUTsT  953 AD-14270  11%  2% cuAuAAuGAAGAGcuuuuuTsT  954 AAAAAGCUCUUcAUuAuAGTsT  955 AD-14271  11%  1% AGAGGAGuGAuAAuuAAAGTsT  956 CUUuAAUuAUcACUCCUCUTsT  957 AD-14272   7%  1% uuucucuGuuAcAAuAcAuTsT  958 AUGuAUUGuAAcAGAGAAATsT  959 AD-14273  14%  2% AAcAucuAuAAuuGcAAcATsT  960 UGUUGcAAUuAuAGAUGUUTsT  961 AD-14274  73%  4% uGcuAGAAGuAcAuAAGAcTsT  962 GUCUuAUGuACUUCuAGcATsT  963 AD-14275  10%  1% AAuGuAcucAAGAcuGAucTsT  964 GAUcAGUCUUGAGuAcAUUTsT  965 AD-14276  89%  2% GuAcucAAGAcuGAucuucTsT  966 GAAGAUcAGUCUUGAGuACTsT  967 AD-14277   7%  1% cAcucuGAuAAAcucAAuGTsT  968 cAUUGAGUUuAUcAGAGUGTsT  969 AD-14278  12%  1% AAGAGcAGAuuAccucuGcTsT  970 GcAGAGGuAAUCUGCUCUUTsT  971 AD-14279 104%  3% ucuGcGAGcccAGAucAAcTsT  972 GUUGAUCUGGGCUCGcAGATsT  973 AD-14280  21%  2% AAcuuGAGccuuGuGuAuATsT  974 uAuAcAcAAGGCUcAAGUUTsT  975 AD-14281  43%  3% GAAuAuAuAuAucAGccGGTsT  976 CCGGCUGAuAuAuAuAUUCTsT  977 AD-14282  45%  6% uGucAucccuAuAGuucAcTsT  978 GUGAACuAuAGGGAUGAcATsT  979 AD-14283  35%  5% GAucuGGcAAccAuAuuucTsT  980 GAAAuAUGGUUGCcAGAUCTsT  981 AD-14284  58%  3% uGGcAAccAuAuuucuGGATsT  982 UCcAGAAAuAUGGUUGCcATsT  983 AD-14285  48%  3% GAuGuuuAccGAAGuGuuGTsT  984 cAAcACUUCGGuAAAcAUCTsT  985 AD-14286  49%  3% uuccuuAucGAGAAucuAATsT  986 UuAGAUUCUCGAuAAGGAATsT  987 AD-14287   6%  1% AGcuuAAuuGcuuucuGGATsT  988 UCcAGAAAGcAAUuAAGCUTsT  989 AD-14288  50%  2% uuGcuAuuAuGGGAGAccATsT  990 UGGUCUCCcAuAAuAGcAATsT  991 AD-14289  48%  1% GucAuGGcGucGcAGccAATsT  992 UUGGCUGCGACGCcAUGACTsT  993 AD-14290 112%  7% uAAuuGcAcuAucuuuGcGTsT  994 CGcAAAGAuAGUGcAAUuATsT  995 AD-14291  77%  2% cuAucuuuGcGuAuGGccATsT  996 UGGCcAuACGcAAAGAuAGTsT  997 AD-14292  80%  6% ucccuAuAGuucAcuuuGuTsT  998 AcAAAGUGAACuAuAGGGATsT  999 AD-14293  58%  2% ucAAccuuuAAuucAcuuGTsT 1000 cAAGUGAAUuAAAGGUUGATsT 1001 AD-14294  77%  2% GGcAAccAuAuuucuGGAATsT 1002 UUCcAGAAAuAUGGUUGCCTsT 1003 AD-14295  62%  2% AuGuAcucAAGAcuGAucuTsT 1004 AGAUcAGUCUUGAGuAcAUTsT 1005 AD-14296  59%  4% GcAGAccAuuuAAuuuGGcTsT 1006 GCcAAAUuAAAUGGUCUGCTsT 1007 AD-14297  37%  1% ucuGAGAGAcuAcAGAuGuTsT 1008 AcAUCUGuAGUCUCUcAGATsT 1009 AD-14298  21%  1% uGcucAuAGAGcAAAGAAcTsT 1010 GUUCUUUGCUCuAUGAGcATsT 1011 AD-14299   6%  1% AcAuAAGAccuuAuuuGGuTsT 1012 ACcAAAuAAGGUCUuAUGUTsT 1013 AD-14300  17%  2% uuuGuGcuGAuucuGAuGGTsT 1014 CcAUcAGAAUcAGcAcAAATsT 1015 AD-14301  97%  6% ccAucAAcAcuGGuAAGAATsT 1016 UUCUuACcAGUGUUGAUGGTsT 1017 AD-14302  13%  1% AGAcAAuuccGGAuGuGGATsT 1018 UCcAcAUCCGGAAUUGUCUTsT 1019 AD-14303  13%  3% GAAcuuGAGccuuGuGuAuTsT 1020 AuAcAcAAGGCUcAAGUUCTsT 1021 AD-14304  38%  2% uAAuuuGGcAGAGcGGAAATsT 1022 UUUCCGCUCUGCcAAAUuATsT 1023 AD-14305  14%  2% uGGAuGAAGuuAuuAuGGGTsT 1024 CCcAuAAuAACUUcAUCcATsT 1025 AD-14306  22%  4% AucuAcAuGAAcuAcAAGATsT 1026 UCUUGuAGUUcAUGuAGAUTsT 1027 AD-14307  26%  6% GGuAuuuuuGAucuGGcAATsT 1028 UUGCcAGAUcAAAAAuACCTsT 1029 AD-14308  62%  8% cuAAuGAAGAGuAuAccuGTsT 1030 cAGGuAuACUCUUcAUuAGTsT 1031 AD-14309  52%  5% uuuGAGAAAcuuAcuGAuATsT 1032 uAUcAGuAAGUUUCUcAAATsT 1033 AD-14310  32%  3% cGAuAAGAuAGAAGAucAATsT 1034 UUGAUCUUCuAUCUuAUCGTsT 1035 AD-14311  23%  2% cuGGcAAccAuAuuucuGGTsT 1036 CcAGAAAuAUGGUUGCcAGTsT 1037 AD-14312  49%  6% uAGAuAccAuuAcuAcAGuTsT 1038 ACUGuAGuAAUGGuAUCuATsT 1039 AD-14313  69%  4% GuAuuAAAuuGGGuuucAuTsT 1040 AUGAAACCcAAUUuAAuACTsT 1041 AD-14314  52%  3% AAGAccuuAuuuGGuAAucTsT 1042 GAUuACcAAAuAAGGUCUUTsT 1043 AD-14315  66%  4% GcuGuuGAuAAGAGAGcucTsT 1044 GAGCUCUCUuAUcAAcAGCTsT 1045 AD-14316  19%  4% uAcucAuGuuucucAGAuuTsT 1046 AAUCUGAGAAAcAUGAGuATsT 1047 AD-14317  16%  5% cAGAuGGAcGuAAGGcAGcTsT 1048 GCUGCCUuACGUCcAUCUGTsT 1049 AD-14318  52% 11% uAucccAAcAGGuAcGAcATsT 1050 UGUCGuACCUGUUGGGAuATsT 1051 AD-14319  28% 11% cAuuGcuAuuAuGGGAGAcTsT 1052 GUCUCCcAuAAuAGcAAUGTsT 1053 AD-14320  52% 10% cccucAGuAAAuccAuGGuTsT 1054 ACcAUGGAUUuACUGAGGGTsT 1055 AD-14321  53%  6% GGucAuuAcuGcccuuGuATsT 1056 uAcAAGGGcAGuAAUGACCTsT 1057 AD-14322  20%  2% AAccAcucAAAAAcAuuuGTsT 1058 cAAAUGUUUUUGAGUGGUUTsT 1059 AD-14323 116%  6% uuuGcAAGuuAAuGAAucuTsT 1060 AGAUUcAUuAACUUGcAAATsT 1061 AD-14324  14%  2% uuAuuuucAGuAGucAGAATsT 1062 UUCUGACuACUGAAAAuAATsT 1063 AD-14325  50%  2% uuuucucGAuucAAAucuuTsT 1064 AAGAUUuGAAUCGAGAAAATsT 1065 AD-14326  47%  3% GuAcGAAAAGAAGuuAGuGTsT 1066 cACuAACUUCUUUUCGuACTsT 1067 AD-14327  18%  2% uuuAAAAcGAGAucuuGcuTsT 1068 AGcAAGAUCUCGUUUuAAATsT 1069 AD-14328  19%  1% GAAuuGAuuAAuGuAcucATsT 1070 UGAGuAcAUuAAUcAAUUCTsT 1071 AD-14329  94% 10% GAuGGAcGuAAGGcAGcucTsT 1072 GAGCUGCCUuACGUCcAUCTsT 1073 AD-14330  60%  4% cAucuGAcuAAuGGcucuGTsT 1074 cAGAGCcAUuAGUcAGAUGTsT 1075 AD-14331  54%  7% GuGAuccuGuAcGAAAAGATsT 1076 UCUUUUCGuAcAGGAUcACTsT 1077 AD-14332  22%  4% AGcucuuAuuAAGGAGuAuTsT 1078 AuACUCCUuAAuAAGAGCUTsT 1079 AD-14333  70% 10% GcucuuAuuAAGGAGuAuATsT 1080 uAuACUCCUuAAuAAGAGCTsT 1081 AD-14334  18%  3% ucuuAuuAAGGAGuAuAcGTsT 1082 CGuAuACUCCUuAAuAAGATsT 1083 AD-14335  38%  6% uAuuAAGGAGuAuAcGGAGTsT 1084 CUCCGuAuACUCCUuAAuATsT 1085 AD-14336  16%  3% cuGcAGcccGuGAGAAAAATsT 1086 UUUUUCUcACGGGCUGcAGTsT 1087 AD-14337  65%  4% ucAAGAcuGAucuucuAAGTsT 1088 CUuAGAAGAUcAGUCUUGATsT 1089 AD-14338  18%  0% cuucuAAGuucAcuGGAAATsT 1090 UUUCcAGUGAACUuAGAAGTsT 1091 AD-14339  20%  4% uGcAAGuuAAuGAAucuuuTsT 1092 AAAGAUUcAUuAACUUGcATsT 1093 AD-14340  24%  1% AAucuAAGGAuAuAGucAATsT 1094 UUGACuAuAUCCUuAGAUUTsT 1095 AD-14341  27%  3% AucucuGAAcAcAAGAAcATsT 1096 UGUUCUUGUGUUcAGAGAUTsT 1097 AD-14342  13%  1% uucuGAAcAGuGGGuAucuTsT 1098 AGAuACCcACUGUUcAGAATsT 1099 AD-14343  19%  1% AGuuAuuuAuAcccAucAATsT 1100 UUGAUGGGuAuAAAuAACUTsT 1101 AD-14344  23%  2% AuGcuAAAcuGuucAGAAATsT 1102 UUUCUGAAcAGUUuAGcAUTsT 1103 AD-14345  21%  4% cuAcAGAGcAcuuGGuuAcTsT 1104 GuAACcAAGUGCUCUGuAGTsT 1105 AD-14346  18%  2% uAuAuAucAGccGGGcGcGTsT 1106 CGCGCCCGGCUGAuAuAuATsT 1107 AD-14347  67%  2% AuGuAAAuAcGuAuuucuATsT 1108 uAGAAAuACGuAUUuAcAUTsT 1109 AD-14348  39%  3% uuuuucucGAuucAAAucuTsT 1110 AGAUUuGAAUCGAGAAAAATsT 1111 AD-14349  83%  6% AAucuuAAcccuuAGGAcuTsT 1112 AGUCCuAAGGGUuAAGAUUTsT 1113 AD-14350  54%  2% ccuuAGGAcucuGGuAuuuTsT 1114 AAAuACcAGAGUCCuAAGGTsT 1115 AD-14351  57%  8% AAuAAAcuGcccucAGuAATsT 1116 UuACUGAGGGcAGUUuAUUTsT 1117 AD-14352  82%  3% GAuccuGuAcGAAAAGAAGTsT 1118 CUUCUUUUCGuAcAGGAUCTsT 1119 AD-14353   2%  1% AAuGuGAuccuGuAcGAAATsT 1120 UUUCGuAcAGGAUcAcAUUTsT 1121 AD-14354  18% 11% GuGAAAAcAuuGGccGuucTsT 1122 GAACGGCcAAUGUUUUcACTsT 1123 AD-14355   2%  1% cuuGAGGAAAcucuGAGuATsT 1124 uACUcAGAGUUUCCUcAAGTsT 1125 AD-14356   8%  2% cGuuuAAAAcGAGAucuuGTsT 1126 cAAGAUCUCGUUUuAAACGTsT 1127 AD-14357   6%  3% uuAAAAcGAGAucuuGcuGTsT 1128 cAGcAAGAUCUCGUUUuAATsT 1129 AD-14358  98% 17% AAAGAuGuAucuGGucuccTsT 1130 GGAGACcAGAuAcAUCUUUTsT 1131 AD-14359  10%  1% cAGAAAAuGuGucuAcucATsT 1132 UGAGuAGAcAcAUUUUCUGTsT 1133 AD-14360   6%  4% cAGGAAuuGAuuAAuGuAcTsT 1134 GuAcAUuAAUcAAUUCCUGTsT 1135 AD-14361  30%  5% AGucAAcuAAAGcAuAuuuTsT 1136 AAAuAUGCUUuAGUUGACUTsT 1137 AD-14362  28%  2% uGuGuAAcAAucuAcAuGATsT 1138 UcAUGuAGAUUGUuAcAcATsT 1139 AD-14363  60%  6% AuAccAuuuGuuccuuGGuTsT 1140 ACcAAGGAAcAAAUGGuAUTsT 1141 AD-14364  12%  9% GcAGAAAucuAAGGAuAuATsT 1142 uAuAUCCUuAGAUUUCUGCTsT 1143 AD-14365   5%  2% uGGcuucucAcAGGAAcucTsT 1144 GAGUUCCUGUGAGAAGCcATsT 1145 AD-14366  28%  5% GAGAuGuGAAucucuGAAcTsT 1146 GUUcAGAGAUUcAcAUCUCTsT 1147 AD-14367  42%  4% uGuAAGccAAuGuuGuGAGTsT 1148 CUcAcAAcAUUGGCUuAcATsT 1149 AD-14368  93% 12% AGccAAuGuuGuGAGGcuuTsT 1150 AAGCCUcAcAAcAUUGGCUTsT 1151 AD-14369  65%  4% uuGuGAGGcuucAAGuucATsT 1152 UGAACUUGAAGCCUcAcAATsT 1153 AD-14370   5%  2% AGGcAGcucAuGAGAAAcATsT 1154 UGUUUCUcAUGAGCUGCCUTsT 1155 AD-14371  54%  5% AuAAAuuGAuAGcAcAAAATsT 1156 UUUUGUGCuAUcAAUUuAUTsT 1157 AD-14372   4%  1% AcAAAAucuAGAAcuuAAuTsT 1158 AUuAAGUUCuAGAUUUUGUTsT 1159 AD-14373   5%  1% GAuAucccAAcAGGuAcGATsT 1160 UCGuACCUGUUGGGAuAUCTsT 1161 AD-14374  92%  6% AAGuuAuuuAuAcccAucATsT 1162 UGAUGGGuAuAAAuAACUUTsT 1163 AD-14375  76%  4% uGuAAAuAcGuAuuucuAGTsT 1164 CuAGAAAuACGuAUUuAcATsT 1165 AD-14376  70%  5% ucuAGuuuucAuAuAAAGuTsT 1166 ACUUuAuAUGAAAACuAGATsT 1167 AD-14377  48%  4% AuAAAGuAGuucuuuuAuATsT 1168 uAuAAAAGAACuACUUuAUTsT 1169 AD-14378  48%  3% ccAuuuGuAGAGcuAcAAATsT 1170 UUUGuAGCUCuAcAAAUGGTsT 1171 AD-14379  44%  5% uAuuuucAGuAGucAGAAuTsT 1172 AUUCUGACuACUGAAAAuATsT 1173 AD-14380  35% 16% AAAucuAAcccuAGuuGuATsT 1174 uAcAACuAGGGUuAGAUUUTsT 1175 AD-14381  44%  5% cuuuAGAGuAuAcAuuGcuTsT 1176 AGcAAUGuAuACUCuAAAGTsT 1177 AD-14382  28%  1% AucuGAcuAAuGGcucuGuTsT 1178 AcAGAGCcAUuAGUcAGAUTsT 1179 AD-14383  55% 11% cAcAAuGAuuuAAGGAcuGTsT 1180 cAGUCCUuAAAUcAUUGUGTsT 1181 AD-14384  48%  9% ucuuuuucucGAuucAAAuTsT 1182 AUUuGAAUCGAGAAAAAGATsT 1183 AD-14385  36%  2% cuuuuucucGAuucAAAucTsT 1184 GAUUuGAAUCGAGAAAAAGTsT 1185 AD-14386  41%  7% AuuuucuGcucAcGAuGAGTsT 1186 CUcAUCGUGAGcAGAAAAUTsT 1187 AD-14387  38%  3% uuucuGcucAcGAuGAGuuTsT 1188 AACUcAUCGUGAGcAGAAATsT 1189 AD-14388  50%  4% AGAGcuAcAAAAccuAuccTsT 1190 GGAuAGGUUUUGuAGCUCUTsT 1191 AD-14389  98%  6% GAGccAAAGGuAcAccAcuTsT 1192 AGUGGUGuACCUUUGGCUCTsT 1193 AD-14390  43%  8% GccAAAGGuAcAccAcuAcTsT 1194 GuAGUGGUGuACCUUUGGCTsT 1195 AD-14391  48%  4% GAAcuGuAcucuucucAGcTsT 1196 GCUGAGAAGAGuAcAGUUCTsT 1197 AD-14392  44%  3% AGGuAAAuAucAccAAcAuTsT 1198 AUGUUGGUGAuAUUuACCUTsT 1199 AD-14393  37%  2% AGcuAcAAAAccuAuccuuTsT 1200 AAGGAuAGGUUUUGuAGCUTsT 1201 AD-14394 114%  7% uGuGAAAGcAuuuAAuuccTsT 1202 GGAAUuAAAUGCUUUcAcATsT 1203 AD-14395  55%  4% GcccAcuuuAGAGuAuAcATsT 1204 UGuAuACUCuAAAGUGGGCTsT 1205 AD-14396  49%  5% uGuGccAcAcuccAAGAccTsT 1206 GGUCUUGGAGUGUGGcAcATsT 1207 AD-14397  71%  6% AAAcuAAAuuGAucucGuATsT 1208 uACGAGAUcAAUUuAGUUUTsT 1209 AD-14398  81%  7% uGAucucGuAGAAuuAucuTsT 1210 AGAuAAUUCuACGAGAUcATsT 1211 AD-14399  38%  4% GcGuGcAGucGGuccuccATsT 1212 UGGAGGACCGACUGcACGCTsT 1213 AD-14400 106%  8% AAAGuuuAGAGAcAucuGATsT 1214 UcAGAUGUCUCuAAACUUUTsT 1215 AD-14401  47%  3% cAGAAGGAAuAuGuAcAAATsT 1216 UUUGuAcAuAUUCCUUCUGTsT 1217 AD-14402  31%  1% cGcccGAGAGuAccAGGGATsT 1218 UCCCUGGuACUCUCGGGCGTsT 1219 AD-14403 105%  4% cGGAGGAGAuAGAAcGuuuTsT 1220 AAACGUUCuAUCUCCUCCGTsT 1221 AD-14404   3%  1% AGAuAGAAcGuuuAAAAcGTsT 1222 CGUUUuAAACGUUCuAUCUTsT 1223 AD-14405  15%  1% GGAAcAGGAAcuucAcAAcTsT 1224 GUuGuGAAGUUCCuGUUCCTsT 1225 AD-14406  44%  5% GuGAGccAAAGGuAcAccATsT 1226 UGGUGuACCUUUGGCUcACTsT 1227 AD-14407  41%  4% AuccucccuAGAcuucccuTsT 1228 AGGGAAGUCuAGGGAGGAUTsT 1229 AD-14408 104%  3% cAcAcuccAAGAccuGuGcTsT 1230 GcAcAGGUCUUGGAGUGUGTsT 1231 AD-14409  67%  4% AcAGAAGGAAuAuGuAcAATsT 1232 UUGuAcAuAUUCCUUCUGUTsT 1233 AD-14410  22%  1% uuAGAGAcAucuGAcuuuGTsT 1234 cAAAGUcAGAUGUCUCuAATsT 1235 AD-14411  29%  3% AAuuGAucucGuAGAAuuATsT 1236 uAAUUCuACGAGAUcAAUUTsT 1237 AD-14412  31%  4%

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 4.

TABLE 4 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a 2′-deoxy-adenosine-5′-phosphate, adenosine-5′- phosphate C, c 2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g 2′-deoxy-guanosine-5′-phosphate, guanosine-5′- phosphate T, t 2′-deoxy-thymidine-5′-phosphate, thymidine-5′- phosphate U, u 2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate N, n any 2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am 2′-O-methyladenosine-5′-phosphate Cm 2′-O-methylcytidine-5′-phosphate Gm 2′-O-methylguanosine-5′-phosphate Tm 2′-O-methyl-thymidine-5′-phosphate Um 2′-O-methyluridine-5′-phosphate Af 2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf 2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf 2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf 2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf 2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, a, underlined: nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined: 2-O-methyl-nucleoside-5′-phosphorothioate um ^(a)capital letters represent 2′-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)

dsRNA Expression Vectors

In another aspect of the invention, Eg5 specific dsRNA molecules that modulate Eg5 gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D 1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single Eg5 gene or multiple Eg5 genes over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection. of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

The Eg5 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Eg5 siRNA In Vitro Screening Via Cell Proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, et al[2002] Biotechniques 33: 1244-8), a cell viability assay was used for siRNA activity screening. HeLa cells (14000 per well [Screens 1 and 3] or 10000 per well [Screen2])) were seeded in 96-well plates and simultaneously transfected with Lipofectamine 2000 (Invitrogen) at a final siRNA concentration in the well of 30 nM and at final concentrations of 50 nM (1^(st) screen) and 25 nM (2^(nd) screen). A subset of duplexes was tested at 25 nM in a third screen (Table 5).

Seventy-two hours post-transfection, cell proliferation was assayed the addition of WST-1 reagent (Roche) to the culture medium, and subsequent absorbance measurement at 450 nm. The absorbance value for control (non-transfected) cells was considered 100 percent, and absorbances for the siRNA transfected wells were compared to the control value. Assays were performed in sextuplicate for each of three screens. A subset of the siRNAs was further tested at a range of siRNA concentrations. Assays were performed in HeLa cells (14000 per well; method same as above, Table 5).

TABLE 5 Relative absorbance at 450 nm Screen I Screen II Screen III Duplex mean sd Mean sd mean Sd AL-DP-6226 20 10 28 11 43 9 AL-DP-6227 66 27 96 41 108 33 AL-DP-6228 56 28 76 22 78 18 AL-DP-6229 17 3 31 9 48 13 AL-DP-6230 48 8 75 11 73 7 AL-DP-6231 8 1 21 4 41 10 AL-DP-6232 16 2 37 7 52 14 AL-DP-6233 31 9 37 6 49 12 AL-DP-6234 103 40 141 29 164 45 AL-DP-6235 107 34 140 27 195 75 AL-DP-6236 48 12 54 12 56 12 AL-DP-6237 73 14 108 18 154 37 AL-DP-6238 64 9 103 10 105 24 AL-DP-6239 9 1 20 4 31 11 AL-DP-6240 99 7 139 16 194 43 AL-DP-6241 43 9 54 12 66 19 AL-DP-6242 6 1 15 7 36 8 AL-DP-6243 7 2 19 5 33 13 AL-DP-6244 7 2 19 3 37 13 AL-DP-6245 25 4 45 10 58 9 AL-DP-6246 34 8 65 10 66 13 AL-DP-6247 53 6 78 14 105 20 AL-DP-6248 7 0 22 7 39 12 AL-DP-6249 36 8 48 13 61 7

The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were re-tested at a range of siRNA concentrations in HeLa cells. The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM. Assays were performed in sextuplicate, and the concentration of each siRNA resulting in fifty percent inhibition of cell proliferation (IC₅₀) was calculated. This dose-response analysis was performed between two and four times for each duplex. Mean IC₅₀ values (nM) are given in Table 6.

TABLE 6 Duplex Mean IC50 AL-DP-6226 15.5 AL-DP-6229 3.4 AL-DP-6231 4.2 AL-DP-6232 17.5 AL-DP-6239 4.4 AL-DP-6242 5.2 AL-DP-6243 2.6 AL-DP-6244 8.3 AL-DP-6248 1.9

Eg5 siRNA In Vitro Screening Via Cell Proliferation

Directly before transfection, Hela S3 (ATCC-Number: CCL-2.2, LCG Promochem GmbH, Wesel, Germany) cells were seeded at 1.5×10⁴ cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium (Ham's F12, 10% fetal calf serum, 100 u penicillin/100 μg/ml streptomycin, all from Biochrom AG, Berlin, Germany). Transfections were performed in quadruplicates. For each well 0.5 μl Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For the siRNA concentration being 50 nM in the 100 μl transfection volume, 1 μl of a 5 μM siRNA were mixed with 11.5 μl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. siRNA-Lipofectamine2000-complexes were applied completely (25 μl each per well) to the cells and cells were incubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau). The single dose screen was done once at 50 nM and at 25 nM, respectively.

Cells were harvested by applying 50 μl of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well containing 100 μl of growth medium and were lysed at 53° C. for 30 min. Afterwards, 50 μl of the lysates were incubated with probesets specific to human Eg5 and human GAPDH and proceeded according to the manufacturer's protocol for QuantiGene. In the end chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the hEg5 probeset were normalized to the respective GAPDH values for each well. Values obtained with siRNAs directed against Eg5 were related to the value obtained with an unspecific siRNA (directed against HCV) which was set to 100% (Tables 1, 2 and 3).

Effective siRNAs from the screen were further characterized by dose response curves. Transfections of dose response curves were performed at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5 μl according to the above protocol. Data analysis was performed by using the Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and applying the dose response model number 205 (Tables 1, 2 and 3).

The lead siRNA AD12115 was additionally analyzed by applying the WST-proliferation assay from Roche (as previously described).

A subset of 34 duplexes from Table 2 that showed greatest activity was assayed by transfection in HeLa cells at final concentrations ranging from 100 nM to 10 fM. Transfections were performed in quadruplicate. Two dose-response assays were performed for each duplex. The concentration giving 20% (IC20), 50% (IC50) and 80% (IC80) reduction of KSP mRNA was calculated for each duplex. (Table 7).

TABLE 7 Concentrations given in pM IC20s IC50s IC80s Duplex 1^(st) 2^(nd) 1st 2nd 1st 2nd name screen screen screen screen screen screen AD12077 1.19 0.80 6.14 10.16 38.63 76.16 AD12078 25.43 25.43 156.18 156.18 ND ND AD12085 9.08 1.24 40.57 8.52 257.68 81.26 AD12095 1.03 0.97 9.84 4.94 90.31 60.47 AD12113 4.00 5.94 17.18 28.14 490.83 441.30 AD12115 0.60 0.41 3.79 3.39 23.45 23.45 AD12125 31.21 22.02 184.28 166.15 896.85 1008.11 AD12134 2.59 5.51 17.87 22.00 116.36 107.03 AD12149 0.72 0.50 4.51 3.91 30.29 40.89 AD12151 0.53 6.84 4.27 10.72 22.88 43.01 AD12152 155.45 7.56 867.36 66.69 13165.27 ND AD12157 0.30 26.23 14.60 92.08 14399.22 693.31 AD12166 0.20 0.93 3.71 3.86 46.28 20.59 AD12180 28.85 28.85 101.06 101.06 847.21 847.21 AD12185 2.60 0.42 15.55 13.91 109.80 120.63 AD12194 2.08 1.11 5.37 5.09 53.03 30.92 AD12211 5.27 4.52 11.73 18.93 26.74 191.07 AD12257 4.56 5.20 21.68 22.75 124.69 135.82 AD12280 2.37 4.53 6.89 20.23 64.80 104.82 AD12281 8.81 8.65 19.68 42.89 119.01 356.08 AD12282 7.71 456.42 20.09 558.00 ND ND AD12285 ND 1.28 57.30 7.31 261.79 42.53 AD12292 40.23 12.00 929.11 109.10 ND ND AD12252 0.02 18.63 6.35 68.24 138.09 404.91 AD12275 25.76 25.04 123.89 133.10 1054.54 776.25 AD12266 4.85 7.80 10.00 32.94 41.67 162.65 AD12267 1.39 1.21 12.00 4.67 283.03 51.12 AD12264 0.92 2.07 8.56 15.12 56.36 196.78 AD12268 2.29 3.67 22.16 25.64 258.27 150.84 AD12279 1.11 28.54 23.19 96.87 327.28 607.27 AD12256 7.20 33.52 46.49 138.04 775.54 1076.76 AD12259 2.16 8.31 8.96 40.12 50.05 219.42 AD12276 19.49 6.14 89.60 59.60 672.51 736.72 AD12321 4.67 4.91 24.88 19.43 139.50 89.49 (ND—not determined)

Silencing of Liver Eg5/KSP in Juvenile Rats Following Single-Bolus Administration of LNP01 Formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can be detected in the growing rat liver. Target silencing with a formulated Eg5/KSP siRNA was evaluated in juvenile rats.

KSP Duplex Tested Duplex ID Target Sense Antisense AD6248 Eg5/ AccGAAGuGuu GGAcAAAcAAc KSP GuuuGuccTsT ACUUCGGUTsT (SEQ ID (SEQ ID NO: 1238) NO: 1239)

Methods

Dosing of Animals.

Male, juvenile Sprague-Dawley rats (19 days old) were administered single doses of lipidoid (“LNP01”) formulated siRNA via tail vein injection. Groups of ten animals received doses of 10 milligrams per kilogram (mg/kg) bodyweight of either AD6248 or an unspecific siRNA. Dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Animals were sacrificed two days after siRNA administration. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.

mRNA Measurements.

Levels of Eg5/KSP mRNA were measured in livers from all treatment groups. Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of Eg5/KSP and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for Eg5/KSP were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for Eg5/KSP mRNA are given. Statistical significance (p value) versus the PBS group is shown (ns, not significant [p>0.05]).

Experiment 1

VEGF/GAPDH p value PBS 1.0 ± 0.47 AD6248 10 mg/kg 0.47 ± 0.12  <0.001 unspec 10 mg/kg 1.0 ± 0.26 ns

A statistically significant reduction in liver Eg5/KSP mRNA was obtained following treatment with formulated AD6248 at a dose of 10 mg/kg.

Silencing of Rat Liver VEGF Following Intravenous Infusion of LNP01 Formulated siRNA Duplexes

A “lipidoid” formulation comprising an equimolar mixture of two siRNAs was administered to rats. One siRNA (AD3133) was directed towards VEGF. The other (AD12115) was directed towards Eg5/KSP. Since Eg5/KSP expression is nearly undetectable in the adult rat liver, only VEGF levels were measured following siRNA treatment.

siRNA Duplexes Administered

Duplex ID Target Sense Antisense AD12115 Eg5/KSP ucGAGAAucuA AGUuAGUUuAG AAcuAAcuTsT AUUCUCGATsT (SEQ ID (SEQ ID NO: 1240) NO: 1241) AD3133 VEGF GcAcAuAGGAG AAGCUcAUCUCU AGAuGAGCUsU CCuAuGuGCusG (SEQ ID (SEQ ID NO: 1242) NO: 1243) Key: A,G,C,U-ribonucleotides; c,u-2′-O-Me ribonucleotides; s-phorphorothioate.

Methods

Dosing of Animals.

Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”) formulated siRNA by a two-hour infusion into the femoral vein. Groups of four animals received doses of 5, 10 and 15 milligrams per kilogram (mg/kg) bodyweight of formulated siRNA. Dose level refers to the total amount of siRNA duplex administered in the formulation. A fourth group received phosphate-buffered saline. Animals were sacrificed 72 hours after the end of the siRNA infusion. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.

Formulation Procedure

The lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%.

mRNA Measurements.

Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of VEGF and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for VEGF were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.

Protein Measurements.

Samples of each liver powder (approximately 60 milligrams) were homogenized in 1 ml RIPA buffer. Total protein concentrations were determined using the Micro BCA protein assay kit (Pierce). Samples of total protein from each animal was used to determine VEGF protein levels using a VEGF ELISA assay (R&D systems). Group means were determined and normalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein (rel. VEGF) are shown for each treatment group. Statistical significance (p value) versus the PBS group for each experiment is shown.

VEGF/GAPDH p value rel VEGF p value PBS  1.0 ± 0.17  1.0 ± 0.17  5 mg/kg 0.74 ± 0.12 <0.05 0.23 ± 0.03 <0.001 10 mg/kg 0.65 ± 0.12 <0.005 0.22 ± 0.03 <0.001 15 mg/kg 0.49 ± 0.17 <0.001 0.20 ± 0.04 <0.001

Statistically significant reductions in liver VEGF mRNA and protein were measured at all three siRNA dose levels. 

1. A composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human kinesin family member 11 (Eg5) gene in a cell, wherein the dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence complementary to the sequence provided in an even SEQ ID NO: 2-580 or an odd SEQ ID NO: 583-1257, wherein the first sequence is complementary to the second sequence and wherein the dsRNA is between 15 and 30 base pairs in length.
 2. The composition of claim 1, wherein the dsRNA comprises at least one modified nucleotide.
 3. The composition of claim 2, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
 4. The composition of claim 2, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
 5. The composition of claim 2, wherein the first dsRNA comprises at least one 2′-O-methyl modified ribonucleotide and at least one phosphorothioate.
 6. The composition of claim 1, wherein the composition, upon contact with a cell expressing Eg5, inhibits expression of Eg5 gene by at least 40%.
 7. The composition of claim 1, wherein the dsRNA is 19-21 base pairs in length.
 8. An isolated cell comprising the composition of claim
 1. 9. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of the composition of claim
 1. 10. An isolated cell comprising the vector of claim
 9. 11. A pharmaceutical composition for inhibiting Eg5 gene expression comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
 12. A method for inhibiting Eg5 gene expression in a cell, the method comprising: introducing into the cell the composition of claim 1; and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the Eg5 gene, thereby inhibiting expression of the Eg5 gene in the cell.
 13. A method of treating pathological processes mediated by human Eg5 expression comprising administering to a patient a therapeutically effective amount of the composition of claim
 1. 